Computationally optimized broadly reactive antigens for influenza

ABSTRACT

Described herein is the development of a computationally optimized influenza HA protein that elicits broadly reactive immune response to all H5N1 influenza virus isolates. The optimized HA protein was developed through a series of HA protein alignments, and subsequent generation of consensus sequences, for clade 2 H5N1 influenza virus isolates. The final consensus HA amino acid sequence was reverse translated and optimized for expression in mammalian cells. It is disclosed herein that influenza virus-like particles containing the optimized HA protein are an effective vaccine against H5N1 influenza virus infection in animals.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/403,407, filed Sep. 14, 2010, which is herein incorporated byreference in its entirety.

FIELD

This disclosure concerns an optimized influenza hemagglutinin proteinthat elicits broadly reactive immune responses to H5N1 virus isolatesand its use as a vaccine.

BACKGROUND

Influenza virus is a member of Orthomyxoviridae family. There are threesubtypes of influenza viruses, designated influenza A, influenza B, andinfluenza C. The influenza virion contains a segmented negative-senseRNA genome, which encodes the following proteins: hemagglutinin (HA),neuraminidase (NA), matrix (M1), proton ion-channel protein (M2),nucleoprotein (NP), polymerase basic protein 1 (PB1), polymerase basicprotein 2 (PB2), polymerase acidic protein (PA), and nonstructuralprotein 2 (NS2). The HA, NA, M1, and M2 are membrane associated, whereasNP, PB1, PB2, PA, and NS2 are nucleocapsid associated proteins. The M1protein is the most abundant protein in influenza particles. The HA andNA proteins are envelope glycoproteins, responsible for virus attachmentand penetration of the viral particles into the cell, and the sources ofthe major immunodominant epitopes for virus neutralization andprotective immunity. Both HA and NA proteins are considered the mostimportant components for prophylactic influenza vaccines.

Each year, seasonal influenza causes over 300,000 hospitalizations and36,000 deaths in the U.S. alone (Simonsen et al., Lancet Infect Dis7:658-66, 2007). The emergence of the novel H1N1 influenza virus in 2009demonstrated how quickly a new influenza pandemic can sweep across theworld. The spread of highly pathogenic H5N1 viruses in birds andcoincident infections in humans have raised the concerns that H5N1viruses may cause a new pandemic in humans. Vaccination is an effectivemethod to prevent influenza infection. There are two influenza vaccineapproaches licensed in the United States; the inactivated, split vaccineand the live-attenuated virus vaccine. Inactivated vaccines canefficiently induce humoral immune responses but generally only poorcellular immune responses. Thus, a need exists for a broadly protectiveinfluenza virus vaccine.

SUMMARY

Disclosed herein is the development of an optimized influenza HA proteinthat elicits broadly reactive immune response to H5N1 influenza virusisolates. The optimized HA protein was developed through a series of HAprotein alignments, and subsequent generation of consensus sequences forclade 2 H5N1 influenza virus isolates (FIG. 1). The final consensus HAamino acid sequence was reverse translated and optimized for expressionin mammalian cells. The optimized HA coding sequence is set forth hereinas SEQ ID NO: 1, and the optimized HA protein sequence is set forthherein as SEQ ID NO: 2.

Provided herein is an isolated nucleic acid molecule comprising anucleotide sequence encoding an optimized influenza HA polypeptide,wherein the nucleotide sequence encoding the HA polypeptide is at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% identical to SEQ ID NO: 1. Optimized influenza HA polypeptidesencoded by the nucleic acid molecule, vectors comprising the nucleicacid molecule, and host cells containing the disclosed vectors are alsoprovided herein.

Further provided is an optimized influenza HA polypeptide, wherein theamino acid sequence of the polypeptide is at least 99% identical to SEQID NO: 2. Also provided are fusion proteins comprising the optimized HApolypeptide, virus-like particles (VLPs) containing the optimized HApolypeptides, and compositions comprising the optimized HA polypeptide.

Collections of plasmids are also provided herein. In some embodiments,the collections of plasmids include a plasmid encoding an influenza NA,a plasmid encoding an influenza MA, and a plasmid encoding the optimizedHA protein disclosed herein.

Further provided is a method of eliciting an immune response toinfluenza virus in a subject by administering the optimized influenza HAprotein, fusion proteins containing the optimized influenza HA, or VLPscontaining the optimized influenza HA, as disclosed herein. Alsoprovided is a method of immunizing a subject against influenza virus byadministering to the subject VLPs containing the optimized influenza HAprotein disclosed herein.

The foregoing and other objects and features of the disclosure willbecome more apparent from the following detailed description, whichproceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B: COBRA HA Design. (A) Schematic illustrating the design ofthe COBRA HA molecule. The phylogenetic tree was inferred fromhemagglutinin amino acid sequences using the maximum likelihood methodand clade/sub-clade groupings were identified. Primary consensussequences were generated for each outbreak group. Secondary consensussequences were then generated for each sub-clade using the primarysequences as input. The secondary consensus sequences were then alignedand the resulting consensus, designated COBRA, was generated. (B)Phylogenetic analysis of the COBRA HA. The unrooted phylogenetic treewas inferred from hemagglutinin amino acid sequences from human H5N1infections isolated from 2004 to 2009 and the clade/sub-clade groupingsare indicated. The star represents the COBRA HA sequence relative tohuman H5N1 infections.

FIGS. 2A-2C: COBRA HA Functional Characterization. COBRA HA wastranslated in vitro and the cell culture lysates were analyzed bySDS-PAGE (A). Lane designations: 1) H5N1 recombinant HA; 2) COBRA HA; 3)Expression vector; 4) H5N1 reassortant virus. The COBRA HA (lane 2)migrates at its expected molecular weight confirming expression of thesynthetic protein. COBRA HA VLPs were prepared in various amounts,serially diluted, and incubated with 1% erythrocytes to evaluatereceptor binding (B). HA titer was determined as the last well in whichthe RBCs remained suspended in a lattice structure. COBRA HA and controllentiviral pseudoparticles packaging a CMV-Luc gene were generated inHEK 293T cells and used to infect MDCK cells with or without trypsin(C). Particle fusion was determined by luciferase production by infectedcells.

FIGS. 3A-3F: COBRA HA Mouse Dosing Immunogenicity. BALB/c mice(n=5/group) were vaccinated at 0 and 3 weeks with blood collected at 14to 21 days after each vaccination. Vaccines were formulated at high (1.5μg HA), and low (0.03 μg HA) doses, with and without Imject® alum, anddelivered intramuscularly. Total IgG at week 5 was determined via ELISAfor each vaccine group (A and B). Values represent the geometric meantiter (±95% confidence interval) of log₁₀ transformed endpoint titers.IgG isotypes were evaluated via ELISA for each vaccine group (C and D).Values represent the mean OD₄₅₀ of a 1:200 dilution of serum.Hemagglutination inhibition (HAI) serum antibody titer for each vaccinegroup was determined at week 5 using representative reassortant viruses(E and F). Values represent the geometric mean titer (±95% confidenceinterval) of log₂ transformed titers. The dotted line represents the1:40 titer. Significant differences were determined by two-way ANOVAwith Bonferroni's post-test to evaluate differences between the vaccineformulations for each test antigen. A p value of less than 0.05 wasconsidered significant.

FIGS. 4A-4D: COBRA HA Mouse Dosing Efficacy. BALB/c mice (n=5/group)were vaccinated with COBRA HA VLPs with or without adjuvant. Mice wereinfected with 5×10³ PFU of the highly pathogenic clade 2.2 H5N1 virusA/Whooper Swan/Mongolia/244/2005. Mice were followed to monitor weightloss (A and B) and sickness (C and D). Sickness score was determined byevaluating activity (0=normal, 1=reduced, 2=severely reduced), hunchedback (0=absent, 1=present) and ruffled fur (0=absent, 1=present). Allmock vaccinated mice reached the experimental endpoint and requiredeuthanasia by 6 days post infection.

FIGS. 5A-5B: Mouse Comparison Immunogenicity. BALB/c mice (n=20/group)were vaccinated at 0 and 3 weeks with blood collected at 14 to 21 daysafter each vaccination. Vaccines were formulated at a high dose (3 μgHA) with Imject® alum and delivered intramuscularly. Total IgG at week 5was determined via ELISA for each vaccine group (A). Values representthe geometric mean titer (±95% confidence interval) of log₁₀ transformedendpoint titers. Hemagglutination inhibition (HAI) serum antibody titerfor each vaccine group was determined at week 5 using representativereassortant viruses (B). Values represent the geometric mean titer (±95%confidence interval) of log₂ transformed titers. The dotted linerepresents the 1:40 titer. Significant differences were determined bytwo-way ANOVA with Bonferroni's post-test to evaluate differencesbetween the vaccine formulations for each test antigen. A p value ofless than 0.05 was considered significant.

FIGS. 6A-6B: Mouse Comparison Efficacy. BALB/c mice (n=20/group) werevaccinated with VLPs and adjuvant. Mice were infected with 5×10³ PFU ofthe highly pathogenic clade 2.2 H5N1 virus A/WhooperSwan/Mongolia/244/2005. Mice were followed to monitor weight loss (A)and sickness (B). Sickness score was determined by evaluating activity(0=normal, 1=reduced, 2=severely reduced), hunched back (0=absent,1=present) and ruffled fur (0=absent, 1=present). All mock(adjuvant-only) vaccinated mice reached the experimental endpoint andrequired euthanasia by 6 days post infection.

FIGS. 7A-7B: Ferret Immunogenicity. Ferrets (n=9/group) were vaccinatedwith VLPs (15 mg HA) with Imject® alum at weeks 0 and 3 and serumcollected at week 5. Total IgG at week 5 was determined via ELISA foreach vaccine group (A). Values represent the geometric mean titer (±95%confidence interval) of log₁₀ transformed endpoint titers.Hemagglutination inhibition (HAI) serum antibody titer for each vaccinegroup was determined at week 5 using representative reassortant viruses(B). Values represent the geometric mean titer (±95% confidenceinterval) of log₂ transformed titers. The dotted line represents the1:40 titer. Significant differences were determined by two-way ANOVAwith Bonferroni's post-test to evaluate differences between the vaccineformulations for each test antigen. A p value of less than 0.05 wasconsidered significant.

FIGS. 8A-8E: Ferret Efficacy. Ferrets (n=9/group) were vaccinated withVLPs formulated with adjuvant. Ferrets were challenged with 1×10⁶ PFU ofthe highly pathogenic clade 2.2 H5N1 virus A/WhooperSwan/Mongolia/244/2005. Animals were monitored daily for weight loss(A), survival (B), temperature (C) and clinical symptoms (D). Relativesickness scores were determined by measuring lethargy (0-3), runny nose(0-1), sneezing (0-1), loss of appetite (0-1) and diarrhea (0-1).Animals reaching experimental endpoint were euthanized according toinstitutional guidelines. Nasal washes were collected serially postinfection and virus titers determined via plaque assay (E). Statisticalsignificance was determined using a two-way ANOVA with Bonferroni's posttest. A p value of less than 0.05 was considered significant.

FIG. 9: Phylogenetic diversity of H5N1 influenza. The unrootedphylogenetic tree was inferred from HA amino acid sequences derived from8 to 10 representative isolates in all clades and sub-clades and theCOBRA HA using the maximum likelihood method. Clade/sub-clade clusterswere identified and are indicated in the shaded ovals. The staridentifies where the COBRA antigen is located relative to the variousrepresentative isolates. Sequences were aligned with MUSCLE 3.7 softwareand the alignment was refined by Gblocks 0.91b software. Phylogeny wasdetermined using the maximum likelihood method with PhyML software.Trees were rendered using TreeDyn 198.3 software (Dereeper et al.,Nucleic Acids Res 36:W465-W469, 2008). The NCBI accession numbers forthe HA sequences used in phylogeny inference were obtained through theInfluenza Virus Resource (Bao et al., J. Virol 82:596-601, 2008).

FIGS. 10A-10F: Serology. Total IgG at week 3 (A) and week 6 (B) wasdetermined via ELISA for each vaccine group. Each collected antiserumwas assayed for antibody binding to representative HA molecules fromclade 2.1 (A/Indonesia/5/2005), clade 2.2 (A/WhooperSwan/Mongolia/244/2005), and clade 2.3 (A/Anhui/1/2005). Valuesrepresent the geometric mean titer (±95% confidence interval) oflog_(in) transformed endpoint titers. Statistical significance of theantibody titer data was determined using a two-way analysis of variance(ANOVA) followed by Bonferroni's post-test to analyze differencesbetween each vaccine group for each of the different antigens that weretested (multiparametric). Significance was defined as p<0.05.Statistical analyses were performed with GraphPad Prism software. HAItiter for each vaccine group was determined at week 3 (C) and week 6 (D)using representative H5N1 influenza viruses: clade 2.1(A/Indonesia/5/2005), clade 2.2 (A/Whooper swan/Mongolia/244/2005), andclade 2.3 (A/Anhui/1/2005). Values represent the geometric mean titer(±95% confidence interval) of log₂ transformed titers. The dotted linerepresents the 1:40 titer. Significant differences were determined bytwo-way ANOVA with Bonferroni's post-test to evaluate differencesbetween the vaccine formulations for each test antigen. A p value ofless than 0.05 was considered significant. The number of monkeys thatresponded with a titer greater than 1:40 is listed above each bar.Neutralizing antibody at week 3 (E) and week 6 (F) was determined viamicroneutralization assay for each vaccine group. Values represent thegeometric mean titer (±95% confidence interval).

FIG. 11: HAI serum antibody titers from vaccinated monkeys against apanel of clade 0, 1, 2, 4, and 7 isolates. HAI titer for each vaccinegroup was determined at week 9 using H5N1 influenza viruses. Valuesrepresent the geometric mean titer (±95% confidence interval) of log₂transformed titers. Significant differences were determined by two-wayANOVA with Bonferroni's post-test to evaluate differences between thevaccine formulations for each test antigen. A p value of less than 0.05was considered significant as described in FIG. 10.

FIGS. 12A-12D: Vaccine induced serum antibody responses. BALB/c mice(n=30/group) or Fitch ferrets (n=6/group) were vaccinated at 0 and 3weeks with blood collected 14 to 21 days after each vaccination. TotalIgG after the second vaccination was determined via ELISA for eachvaccine group (A and C). Receptor blocking antibody titers after thesecond vaccination were determined via hemagglutination inhibition (HAI)for each vaccine group (B and D). Values represent the geometric mean ofthe reciprocal dilution (+/−95% confidence interval) of the lastpositive well. Significant differences between COBRA and polyvalentvaccines were determined by a two-tailed Student's T test and a p valueof less than 0.05 was considered significant (*).

FIGS. 13A-13D: Highly pathogenic Clade 2.2 challenge. Vaccinated BALB/cmice (n=5/group) were infected with 5×10³ PFU of the highly pathogenicclade 2.2 H5N1 virus A/Whooper Swan/Mongolia/244/2005 (WS/05). Mice weremonitored daily for weight loss (A) and sickness (B). Vaccinated Fitchferrets (n=6/group) were infected with 1×10⁶ PFU of the highlypathogenic clade WS/05 virus. Ferrets were monitored daily for weightloss (C) and sickness (D). Values represent mean (+/−SEM) for eachgroup.

FIGS. 14A-14B: Clade 2.2 viral loads. Vaccinated BALB/c mice(n=15/group) were infected with 5×10³ PFU of the highly pathogenic clade2.2 H5N1 virus A/Whooper Swan/Mongolia/244/2005 (WS/05). Cohorts of mice(n=5/group) were sacrificed at 1, 3 and 5 days post infection, lungsharvested, and viral load determined by plaque assay (A). VaccinatedFitch ferrets (n=6/group) were infected with 1×10⁶ PFU of the highlypathogenic WS/05 virus. Nasal washes were collected and viral loaddetermined by plaque assay (B). Values represent mean (+/−SEM) viraltiter for each group. Significant differences between COBRA andpolyvalent vaccines were determined by a two-tailed Student's T test anda p value of less than 0.05 was considered significant (*).

FIGS. 15A-15B: Histopathology of infected lungs. Vaccinated BALB/c mice(n=15/group) were infected with 5×10³ PFU of the highly pathogenic clade2.2 H5N1 virus A/Whooper Swan/Mongolia/244/2005 (WS/05). Cohorts of mice(n=5/group) were sacrificed at 3 days post infection and in situhybridization (ISH) for influenza matrix protein (MP) was performed onsections from paraffin embedded lung tissue (A). Severity of influenzaISH foci was accessed in the bronchi (B). Scoring: 0=no definitivesignal; 1=occasional focus; 2=focus in most fields; 3=more than onefocus per field.

FIGS. 16A-16C: Clade 1 challenge. Vaccinated BALB/c mice (n=4/group)were infected with 5×10³ PFU of reassortant virus containing the HA andNA genes from the clade 1 H5N1 virus A/Vietnam/1203/2004 (VN/04). Micewere monitored daily for weight loss (A) and sickness (B). Valuesrepresent mean (+/−SD) for each group. An additional cohort ofvaccinated mice (n=3/group) were infected and lungs were harvested 3days post infection for analysis of viral burden (C). Values representmean (+/−SEM) viral titer for each group.

FIGS. 17A-17B: Post-challenge cellular immune responses. VaccinatedBALB/c mice (n=3/group) were infected with 5×10³ PFU of reassortantvirus containing the HA and NA genes from the clade 1 H5N1 virusA/Vietnam/1203/2004 (VN/04). Mice were sacrificed 6 days post infection,lungs were harvested and the numbers of antibody secreting cells (A) andIFN-γ producing cells (B) were determined by ELISpot assay. Valuesrepresent the mean (+/−SEM) spots for each group.

FIGS. 18A-18B: Passive transfer clade 1 challenge. BALB/c mice(n=10/group) were vaccinated at 0 and 3 weeks with blood collected 14 to21 days after each vaccination. Serum collected after the secondvaccination was pooled for each vaccine group and administered to naïverecipient mice (n=5/group). One day after passive transfer, recipientmice were infected with 5×10³ PFU of reassortant virus containing the HAand NA genes from the clade 1 H5N1 virus A/Vietnam/1203/2004 (VN/04).Mice were monitored daily for weight loss (A) and sickness (B). Valuesrepresent mean (+/−SD) for each group. Significant differences weredetermined by two-way ANOVA with Bonferroni's post-test to evaluatedifferences between vaccines at each day. A p value of less than 0.05was considered significant (*).

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is incorporated by referenceherein. In the accompanying sequence listing:

SEQ ID NOs: 1 and 2 are the nucleotide and amino acid sequences,respectively, of a codon-optimized influenza HA (designated COBRA).

SEQ ID NOs: 3 and 4 are the nucleotide and amino acid sequences,respectively, of a codon-optimized influenza NA.

SEQ ID NOs: 5 and 6 are the nucleotide and amino acid sequences,respectively, of a codon-optimized influenza M1.

SEQ ID NO: 7 is the nucleotide sequence of a plasmid encoding acodon-optimized influenza HA.

SEQ ID NO: 8 is the nucleotide sequence of a plasmid encoding acodon-optimized influenza NA.

SEQ ID NO: 9 is the nucleotide sequence of a plasmid encoding acodon-optimized influenza M1.

SEQ ID NO: 10 is the amino acid sequence of a T cell epitope in H5 HA(HA₅₃₃).

SEQ ID NO: 11 is the amino acid sequence of an ovalbumin T cell epitope(OVa₂₅₇).

DETAILED DESCRIPTION I. Abbreviations

-   -   ASC: antibody secreting cell    -   DPI: days post infection    -   HA: hemagglutinin or hemagglutination assay    -   HAI: hemagglutination inhibition    -   hRBC: horse red blood cell    -   IFU: infectious unit    -   LD₅₀: lethal dose 50    -   M1: matrix protein 1    -   MN: microneutralization    -   MOI: multiplicity of infection    -   NA: neuraminidase    -   PFU: plaque form unit    -   RDE: receptor destroying enzyme    -   TCID: tissue culture infectious dose    -   tRBC: turkey red blood cell    -   VLP: virus-like particle

II. Terms and Methods

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Adjuvant: A substance or vehicle that non-specifically enhances theimmune response to an antigen. Adjuvants can include a suspension ofminerals (alum, aluminum hydroxide, or phosphate) on which antigen isadsorbed; or water-in-oil emulsion in which antigen solution isemulsified in mineral oil (for example, Freund's incomplete adjuvant),sometimes with the inclusion of killed mycobacteria (Freund's completeadjuvant) to further enhance antigenicity. Immunostimulatoryoligonucleotides (such as those including a CpG motif) can also be usedas adjuvants (for example, see U.S. Pat. Nos. 6,194,388; 6,207,646;6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199).Adjuvants also include biological molecules, such as costimulatorymolecules. Exemplary biological adjuvants include IL-2, RANTES, GM-CSF,TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL.

Administer: As used herein, administering a composition to a subjectmeans to give, apply or bring the composition into contact with thesubject. Administration can be accomplished by any of a number ofroutes, such as, for example, topical, oral, subcutaneous,intramuscular, intraperitoneal, intravenous, intrathecal andintramuscular.

Antibody: An immunoglobulin molecule produced by B lymphoid cells with aspecific amino acid sequence. Antibodies are evoked in humans or otheranimals by a specific antigen (immunogen). Antibodies are characterizedby reacting specifically with the antigen in some demonstrable way,antibody and antigen each being defined in terms of the other.“Eliciting an antibody response” refers to the ability of an antigen orother molecule to induce the production of antibodies.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T-cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. In some embodimentsof the disclosed compositions and methods, the antigen is an influenzaHA protein.

Attenuated: In the context of a live virus, the virus is attenuated ifits ability to infect a cell or subject and/or its ability to producedisease is reduced (for example, eliminated) compared to a wild-typevirus. Typically, an attenuated virus retains at least some capacity toelicit an immune response following administration to an immunocompetentsubject. In some cases, an attenuated virus is capable of eliciting aprotective immune response without causing any signs or symptoms ofinfection. In some embodiments, the ability of an attenuated virus tocause disease in a subject is reduced at least about 10%, at least about25%, at least about 50%, at least about 75% or at least about 90%relative to wild-type virus.

Clade: Refers to the different categorizations of the known influenzaviruses, such as influenza A H5N1 viruses. Viruses in an H5N1 clade aregenetically related, but do not share the exact viral genome. There areat least ten different clades of H5N1 subtypes designated in the art:clade 0 clade 1, clade 2, clade 3, clade 4, clade 5, clade 6, clade 7,clade 8 and clade 9 (Abdel-Ghafar et al., N Engl J Med 358:261-273,2008). Clade 2 is further divided into sub-clades (including clade 2.1,clade 2.2, clade 2.3, clade 2.4 and clade 2.5).

Codon-optimized: A “codon-optimized” nucleic acid refers to a nucleicacid sequence that has been altered such that the codons are optimal forexpression in a particular system (such as a particular species of groupof species). For example, a nucleic acid sequence can be optimized forexpression in mammalian cells. Codon optimization does not alter theamino acid sequence of the encoded protein.

Fusion protein: A protein generated by expression of a nucleic acidsequence engineered from nucleic acid sequences encoding at least aportion of two different (heterologous) proteins. To create a fusionprotein, the nucleic acid sequences must be in the same reading frameand contain to internal stop codons. For example, a fusion proteinincludes an influenza HA fused to a heterologous protein.

Geographical location or geographical region: Refers to preselecteddivisions of geographical areas of the earth, for example, by continentor other preselected territory or subdivision (e.g., the Middle East,which spans more than one continent). Examples of different geographicalregions include countries (e.g., Turkey, Egypt, Iraq, Azerbaijan, China,United States), continents (e.g., Asia, Europe, North America, SouthAmerica, Oceania, Africa), and recognized geopolitical subdivisions(such as the Middle East).

Hemagglutinin (HA): An influenza virus surface glycoprotein. HA mediatesbinding of the virus particle to a host cells and subsequent entry ofthe virus into the host cell. The nucleotide and amino acid sequences ofnumerous influenza HA proteins are known in the art and are publicallyavailable, such as those deposited with GenBank (see Table 1 for a listof GenBank Accession Nos. of H5N1 HA sequences). HA (along with NA) isone of the two major influenza virus antigenic determinants.

Immune response: A response of a cell of the immune system, such as aB-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus suchas an antigen or vaccine. An immune response can include any cell of thebody involved in a host defense response, including for example, anepithelial cell that secretes an interferon or a cytokine. An immuneresponse includes, but is not limited to, an innate immune response orinflammation. As used herein, a protective immune response refers to animmune response that protects a subject from infection (preventsinfection or prevents the development of disease associated withinfection). Methods of measuring immune responses are well known in theart and include, for example, measuring proliferation and/or activity oflymphocytes (such as B or T cells), secretion of cytokines orchemokines, inflammation, antibody production and the like.

Immunogen: A compound, composition, or substance which is capable, underappropriate conditions, of stimulating an immune response, such as theproduction of antibodies or a T-cell response in an animal, includingcompositions that are injected or absorbed into an animal. As usedherein, as “immunogenic composition” is a composition comprising animmunogen (such as an HA polypeptide).

Immunize: To render a subject protected from an infectious disease, suchas by vaccination.

Influenza virus: A segmented negative-strand RNA virus that belongs tothe Orthomyxoviridae family. There are three types of Influenza viruses,A, B and C. Influenza A viruses infect a wide variety of birds andmammals, including humans, horses, marine mammals, pigs, ferrets, andchickens. In animals, most influenza A viruses cause mild localizedinfections of the respiratory and intestinal tract. However, highlypathogenic influenza A strains, such as H5N1, cause systemic infectionsin poultry in which mortality may reach 100%. H5N1 is also referred toas “avian influenza.”

Isolated: An “isolated” biological component (such as a nucleic acid,protein or virus) has been substantially separated or purified away fromother biological components (such as cell debris, or other proteins ornucleic acids). Biological components that have been “isolated” includethose components purified by standard purification methods. The termalso embraces recombinant nucleic acids, proteins or viruses, as well aschemically synthesized nucleic acids or peptides.

Linker: One or more amino acids that serve as a spacer between twopolypeptides of a fusion protein.

Matrix (M1) protein: An influenza virus structural protein found withinthe viral envelope. M1 is thought to function in assembly and budding.

Neuraminidase (NA): An influenza virus membrane glycoprotein. NA isinvolved in the destruction of the cellular receptor for the viral HA bycleaving terminal sialic acid residues from carbohydrate moieties on thesurfaces of infected cells. NA also cleaves sialic acid residues fromviral proteins, preventing aggregation of viruses. NA (along with HA) isone of the two major influenza virus antigenic determinants.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Optimized influenza HA protein: As used herein, “optimized influenza HAprotein” refers to the HA protein consensus sequence generated bysequence alignments of clade 2 H5N1 influenza viruses (as described inExample 1 below). The nucleotide sequence encoding the optimized HAprotein was further optimized for expression in mammalian cells viacodon-optimization and RNA optimization (such as to increase RNAstability). The optimized influenza HA protein disclosed herein (and setforth herein as SEQ ID NO: 2) is also referred to as “COBRA.”

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids without any termination codons. These sequencesare usually translatable into a peptide.

Outbreak: As used herein, an influenza virus “outbreak” refers to acollection of virus isolates from within a single country in a givenyear.

Pharmaceutically acceptable vehicles: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 15th Edition (1975), describes compositions andformulations suitable for pharmaceutical delivery of one or moretherapeutic compositions, such as one or more influenza vaccines, andadditional pharmaceutical agents.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Plasmid: A circular nucleic acid molecule capable of autonomousreplication in a host cell.

Polypeptide: A polymer in which the monomers are amino acid residueswhich are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used. The terms “polypeptide” or “protein” as used herein areintended to encompass any amino acid sequence and include modifiedsequences such as glycoproteins. The term “polypeptide” is specificallyintended to cover naturally occurring proteins, as well as those whichare recombinantly or synthetically produced. The term “residue” or“amino acid residue” includes reference to an amino acid that isincorporated into a protein, polypeptide, or peptide.

Conservative amino acid substitutions are those substitutions that, whenmade, least interfere with the properties of the original protein, thatis, the structure and especially the function of the protein isconserved and not significantly changed by such substitutions. Examplesof conservative substitutions are shown below.

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

Conservative substitutions generally maintain (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.

The substitutions which in general are expected to produce the greatestchanges in protein properties will be non-conservative, for instancechanges in which (a) a hydrophilic residue, for example, seryl orthreonyl, is substituted for (or by) a hydrophobic residue, for example,leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, for example, lysyl, arginyl, orhistadyl, is substituted for (or by) an electronegative residue, forexample, glutamyl or aspartyl; or (d) a residue having a bulky sidechain, for example, phenylalanine, is substituted for (or by) one nothaving a side chain, for example, glycine.

Preventing, treating or ameliorating a disease: “Preventing” a diseaserefers to inhibiting the full development of a disease. “Treating”refers to a therapeutic intervention that ameliorates a sign or symptomof a disease or pathological condition after it has begun to develop.“Ameliorating” refers to the reduction in the number or severity ofsigns or symptoms of a disease.

Promoter: An array of nucleic acid control sequences which directtranscription of a nucleic acid. A promoter includes necessary nucleicacid sequences near the start site of transcription. A promoter alsooptionally includes distal enhancer or repressor elements. A“constitutive promoter” is a promoter that is continuously active and isnot subject to regulation by external signals or molecules. In contrast,the activity of an “inducible promoter” is regulated by an externalsignal or molecule (for example, a transcription factor). In someembodiments herein, the promoter is a CMV promoter.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purifiedpeptide, protein, virus, or other active compound is one that isisolated in whole or in part from naturally associated proteins andother contaminants. In certain embodiments, the term “substantiallypurified” refers to a peptide, protein, virus or other active compoundthat has been isolated from a cell, cell culture medium, or other crudepreparation and subjected to fractionation to remove various componentsof the initial preparation, such as proteins, cellular debris, and othercomponents.

Recombinant: A recombinant nucleic acid, protein or virus is one thathas a sequence that is not naturally occurring or has a sequence that ismade by an artificial combination of two otherwise separated segments ofsequence. This artificial combination is often accomplished by chemicalsynthesis or by the artificial manipulation of isolated segments ofnucleic acids, for example, by genetic engineering techniques.

Sequence identity: The similarity between amino acid or nucleic acidsequences is expressed in terms of the similarity between the sequences,otherwise referred to as sequence identity. Sequence identity isfrequently measured in terms of percentage identity (or similarity orhomology); the higher the percentage, the more similar the two sequencesare. Homologs or variants of a given gene or protein will possess arelatively high degree of sequence identity when aligned using standardmethods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higginsand Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research16:10881-10890, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988. Altschul et al., Nature Genet. 6:119-129, 1994.

The NCBI Basic Local Alignment Search Tool (BLAST™) (Altschul et al., J.Mol. Biol. 215:403-410, 1990) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and non-human mammals, such as non-human primates.In one example, a subject is one who is infected with H5N1 or issusceptible to such infection.

Therapeutically effective amount: A quantity of a specified agentsufficient to achieve a desired effect in a subject being treated withthat agent. For example, this may be the amount of an influenza virusvaccine useful for eliciting an immune response in a subject and/or forpreventing infection by influenza virus. Ideally, in the context of thepresent disclosure, a therapeutically effective amount of an influenzavaccine is an amount sufficient to increase resistance to, prevent,ameliorate, and/or treat infection caused by influenza virus in asubject without causing a substantial cytotoxic effect in the subject.The effective amount of an influenza vaccine useful for increasingresistance to, preventing, ameliorating, and/or treating infection in asubject will be dependent on, for example, the subject being treated,the manner of administration of the therapeutic composition and otherfactors.

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

Vaccine: A preparation of immunogenic material capable of stimulating animmune response, administered for the prevention, amelioration, ortreatment of disease, such as an infectious disease. The immunogenicmaterial may include, for example, attenuated or killed microorganisms(such as attenuated viruses), or antigenic proteins, peptides or DNAderived from them. Vaccines may elicit both prophylactic (preventative)and therapeutic responses. Methods of administration vary according tothe vaccine, but may include inoculation, ingestion, inhalation or otherforms of administration. Inoculations can be delivered by any of anumber of routes, including parenteral, such as intravenous,subcutaneous or intramuscular. Vaccines may be administered with anadjuvant to boost the immune response.

Vector: A vector is a nucleic acid molecule allowing insertion offoreign nucleic acid without disrupting the ability of the vector toreplicate and/or integrate in a host cell. A vector can include nucleicacid sequences that permit it to replicate in a host cell, such as anorigin of replication. An insertional vector is capable of insertingitself into a host nucleic acid. A vector can also include one or moreselectable marker genes and other genetic elements. An expression vectoris a vector that contains the necessary regulatory sequences to allowtranscription and translation of inserted gene or genes. In someembodiments of the present disclosure, the vector encodes an influenzaHA, NA or M1 protein. In some embodiments, the vector is the pTR600expression vector (U.S. Patent Application Publication No. 2002/0106798;Ross et al., Nat. Immunol. 1(2):102-103, 2000; Green et al., Vaccine20:242-248, 2001).

Virus-like particle (VLP): Virus particles made up of one of more viralstructural proteins, but lacking the viral genome. Because VLPs lack aviral genome, they are non-infectious. In addition, VLPs can often beproduced by heterologous expression and can be easily purified. MostVLPs comprise at least a viral core protein that drives budding andrelease of particles from a host cell. One example of such a coreprotein is influenza M1. In some embodiments herein, an influenza VLPcomprises the HA, NA and M1 proteins. As described herein, influenzaVLPs can be produced by transfection of host cells with plasmidsencoding the HA, NA and M1 proteins. After incubation of the transfectedcells for an appropriate time to allow for protein expression (such asfor approximately 72 hours), VLPs can be isolated from cell culturesupernatants. Example 1 provides an exemplary protocol for purifyinginfluenza VLPs from cell supernatants. In this example, VLPs areisolated by low speed centrifugation (to remove cell debris), vacuumfiltration and ultracentrifugation through 20% glycerol.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means including A, or B, or A and B. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. All GenBank Accession numbers areincorporated by reference herein as they appeared in the database onSep. 9, 2010. In case of conflict, the present specification, includingexplanations of terms, will control. In addition, the materials,methods, and examples are illustrative only and not intended to belimiting.

III. Overview of Several Embodiments

Disclosed herein is the development of a computationally optimizedinfluenza HA protein that elicits broadly reactive immune responses toH5N1 influenza virus isolates, such as the isolates listed in Table 1.The optimized HA protein was developed through a series of HA proteinalignments, and subsequent generation of consensus sequences, for clade2 H5N1 influenza virus isolates (described in detail in Example 1 below;see also FIG. 1). The final consensus HA amino acid sequence was reversetranslated and optimized for expression in mammalian cells. Optimizationof the nucleic acid sequence included optimization of the codons forexpression in mammalian cells and RNA optimization (such as RNAstability). The optimized HA coding sequence is set forth herein as SEQID NO: 1, and the optimized HA protein sequence is set forth herein asSEQ ID NO: 2.

Thus, provided herein is an isolated nucleic acid molecule comprising anucleotide sequence encoding an influenza HA polypeptide. In someembodiments, the nucleotide sequence encoding the HA polypeptide is atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% identical to SEQ ID NO: 1.

In some examples, the nucleotide sequence encoding the influenza HApolypeptide that is at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to SEQ ID NO: 1 lacks thestart codon (nucleotides 1-3 of SEQ ID NO: 1), encoding a N-terminalmethionine. In particular examples, the nucleotide sequence encoding theinfluenza HA polypeptide is at least 94% identical to nucleotides 4-1707of SEQ ID NO: 1. In other examples, the nucleotide sequence encoding theHA polypeptide comprises or consists of nucleotides 4-1707 of SEQ ID NO:1.

In some examples, the nucleotide sequence encoding the HA polypeptidecomprises SEQ ID NO: 1. In particular examples, the nucleotide sequenceencoding the HA polypeptide consists of SEQ ID NO: 1. Also providedherein are influenza HA polypeptides encoded by the disclosed nucleicacid molecules.

Further provided are vectors containing a nucleotide sequence encodingan optimized HA polypeptide. In some embodiments of the vectors providedherein, the nucleotide sequence encoding the HA polypeptide is at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% identical to SEQ ID NO: 1. In some examples, the vector furtherincludes a promoter operably linked to the nucleotide sequence encodingthe HA polypeptide. In particular examples, the promoter is acytomegalovirus (CMV) promoter. In some embodiments, the nucleotidesequence of the vector is at least 85%, at least 90%, at least 95%, atleast 98% or at least 99% identical to the nucleotide sequence of SEQ IDNO: 7. In some examples, the nucleotide sequence of the vector comprisesthe nucleotide sequence of SEQ ID NO: 7. In particular examples, thenucleotide sequence of the vector consists of the nucleotide sequence ofSEQ ID NO: 7.

Also provided herein are influenza HA polypeptides produced bytransfecting a host cell with a vector provided herein under conditionssufficient to allow for expression of the HA polypeptide. Isolated cellscontaining the disclosed vectors are also provided.

Also provided herein are optimized influenza HA polypeptides. In someembodiments, the amino acid sequence of the polypeptide is at least 99%identical to SEQ ID NO: 2. In some examples, the amino acid sequence ofthe influenza HA polypeptide that is at least 99% identical to SEQ IDNO: 2 lacks the N-terminal methionine residue. In particular examples,the amino acid sequence of the influenza HA polypeptide is at least 99%identical to amino acids 2-568 of SEQ ID NO: 2. In other examples, theamino acid sequence of the HA polypeptides comprises or consists ofamino acids 2-568 of SEQ ID NO: 2.

In some examples, the amino acid sequence of the polypeptide comprisesthe amino acid sequence of SEQ ID NO: 2. In particular examples, theamino acid sequence of the polypeptide consists of the amino acidsequence of SEQ ID NO: 2. Fusion proteins comprising the influenza HApolypeptides disclosed herein are also provided. The influenza HApolypeptide can be fused to any heterologous amino acid sequence to formthe fusion protein.

Further provided herein are influenza virus-like particles (VLPs)containing an optimized influenza HA protein disclosed herein. In someembodiments, the HA protein of the VLP is at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% identical toSEQ ID NO: 2. The influenza VLPs can further include any additionalinfluenza proteins necessary to form the virus particle. In someembodiments, the influenza VLPs further include influenza neuraminidase(NA) protein, influenza matrix (M1) protein, or both.

In some embodiments of the influenza VLPs, the amino acid sequence ofthe influenza NA protein is at least 85%, at least 90%, at least 95%, atleast 98% or at least 99% identical to SEQ ID NO: 4. In some examples,the amino acid sequence of the influenza NA protein comprises SEQ ID NO:4. In particular examples, the amino acid sequence of the influenza NAprotein consists of SEQ ID NO: 4. In some embodiments, the amino acidsequence of the influenza M1 protein is at least 85%, at least 90%, atleast 95%, at least 98% or at least 99% identical to SEQ ID NO: 6. Insome examples, the amino acid sequence of the influenza M1 proteincomprises SEQ ID NO: 6. In particular examples, the amino acid sequenceof the influenza M1 protein consists of SEQ ID NO: 6.

Also provided is an influenza VLP containing an influenza HA polypeptideas described herein, produced by transfecting a host cell with a vectorencoding the HA polypeptide, a vector encoding an influenza NA proteinand a vector encoding an influenza M1 protein, under conditionssufficient to allow for expression of the HA, M1 and NA proteins.

The vectors used to express the HA, NA and M1 proteins can be anysuitable expression vectors known in the art. The vectors can be, forexample, mammalian expression vectors, or viral vectors. In someembodiments, the vector is the pTR600 expression vector (U.S. PatentApplication Publication No. 2002/0106798, herein incorporated byreference; Ross et al., Nat. Immunol. 1(2):102-103, 2000; Green et al.,Vaccine 20:242-248, 2001).

In some embodiments, the nucleotide sequence of the vector encoding theHA polypeptide is at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:7. In some examples, the nucleotide sequence of the vector encoding theHA polypeptide comprises SEQ ID NO: 7. In particular examples, thenucleotide sequence of the vector encoding the HA polypeptide consistsof SEQ ID NO: 7.

In some embodiments, the nucleotide sequence of the vector encoding theNA protein is at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% identical to SEQ ID NO: 8. Insome examples, the nucleotide sequence of the vector encoding the NAprotein comprises SEQ ID NO: 8. In particular examples, the nucleotidesequence of the vector encoding the NA protein consists of SEQ ID NO: 8.

In some embodiments, the nucleotide sequence of the vector encoding theM1 protein is at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% identical to SEQ ID NO: 9. Insome examples, the nucleotide sequence of the vector encoding the M1protein comprises SEQ ID NO: 9. In particular examples, the nucleotidesequence of the vector encoding the M1 protein consists of SEQ ID NO: 9.

Collections of plasmids are also provided herein. In some embodiments,the collection of plasmids includes a plasmid encoding an influenza NA,a plasmid encoding an influenza MA, and a plasmid encoding the optimizedHA protein disclosed herein. In some embodiments, the nucleotidesequence encoding the codon-optimized influenza HA of the HA-encodingplasmid is at least 94%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% identical to SEQ ID NO: 1. Also provided arekits comprising the collection of plasmids.

In some embodiments of the collections of plasmids, the influenza NA iscodon-optimized and/or the influenza M1 is codon-optimized. In someexamples, the nucleotide sequence encoding the codon-optimized influenzaNA is at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98% or at least 99% identical to SEQ ID NO: 3. Inparticular examples, the nucleotide sequence encoding thecodon-optimized influenza NA comprises, or consists of, SEQ ID NO: 3. Insome examples, the nucleotide sequence encoding the codon-optimizedinfluenza M1 is at least 85%, at least 90%, at least 95%, at least 96%,at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 5. Inparticular examples, the nucleotide sequence encoding thecodon-optimized influenza M1 comprises, or consists of, SEQ ID NO: 5.

In one non-limiting example, the plasmid encoding influenza NA comprisesSEQ ID NO: 8; the plasmid encoding influenza M1 comprises SEQ ID NO: 9;and the plasmid encoding influenza HA comprises SEQ ID NO: 10.

In some embodiments, transfection of the collection of plasmids intohost cells under conditions sufficient to allow for expression of theHA, NA and M1 proteins produces influenza VLPs comprising the HA, NA andM1 proteins.

Also provided herein are compositions comprising an optimized influenzaHA protein as disclosed herein, or a fusion protein or VLP comprisingthe optimized influenza HA protein. In some embodiments, thecompositions further comprise a pharmaceutically acceptable carrierand/or an adjuvant. For example, the adjuvant can be alum, Freund'scomplete adjuvant, a biological adjuvant or immunostimulatoryoligonucleotides (such as CpG oligonucleotides).

Further provided is a method of eliciting an immune response toinfluenza virus in a subject by administering an influenza HA proteindisclosed herein, fusion proteins containing the influenza HA, or VLPscontaining the influenza HA, as disclosed herein. In some embodiments,the influenza virus is an H5N1 influenza virus. In some embodiments, theHA protein, HA fusion protein or VLP can be administered using anysuitable route of administration, such as, for example, intramuscular.In some embodiments, the HA protein, fusion protein or VLP isadministered as a composition further comprising a pharmaceuticallyacceptable carrier and/or an adjuvant. For example, the adjuvant can bealum. Freund's complete adjuvant, a biological adjuvant orimmunostimulatory oligonucleotides (such as CpG oligonucleotides).

Also provided is a method of immunizing a subject against influenzavirus by administering to the subject VLPs containing the optimizedinfluenza HA protein disclosed herein, or administering a compositionthereof. In some embodiments of the method, the composition furthercomprises a pharmaceutically acceptable carrier and/or an adjuvant. Forexample, the adjuvant can be alum, Freund's complete adjuvant, abiological adjuvant or immunostimulatory oligonucleotides (such as CpGoligonucleotides). In some embodiments, the VLPs (or compositionsthereof) are administered intramuscularly.

In some embodiments of the methods of eliciting an immune response orimmunizing a subject, the subject is administered at least 1 μg of theVLPs containing the optimized HA protein, such as at least 5 μg, atleast 10 μg, at least 15 μg, at least 20 μg, at least 25 μg, at least 30μg, at least 40 μg or at least 50 μg of the VLPs containing theoptimized HA protein, for example about 1 to about 50 μg or about 1 toabout 25 μg of the VLPs containing the optimized HA protein. Inparticular examples, the subject is administered about 5 to about 20 μgof the VLPs, or about 10 to about 15 μg of the VLPs. In one specificnon-limiting example, the subject is administered about 15 μg of theVLPs. However, one of skill in the art is capable of determining atherapeutically effective amount (for example an amount that providesprotection against H5N1 influenza virus infection) of VLPs to administerto a subject.

It is disclosed herein that administration of VLPs comprising the COBRAHA disclosed herein (SEQ ID NO: 2) elicits protective levels of HAIantibodies to a number of representative clade 2 isolates and providescomplete protection against lethal challenge with a clade 2.2 H5N1virus. In some embodiments, administration of VLPs containing anoptimized influenza HA results in production of high HAI titers (≧1:40)to H5N1 clade 1, clade 2.1, clade 2.2 and clade 2.3 isolates. In someexamples, the VLPs containing an optimized influenza HA elicit high HAItiters against clade 1 and/or clade 7 viruses. The VLPs containing anoptimized influenza HA disclosed herein elicit a broader immune response(e.g., elicit neutralizing antibodies to a broader range is H5N1 virusisolates, such as those from clade 1, sub-clades of clade 2, and clade7) than a polyvalent influenza virus vaccine.

Also provided herein is a method of optimizing an influenza proteinsequence to elicit broadly reactive immune responses in a subject. Inthe context of the present disclosure, “broadly reactive” means theprotein sequence elicits an immune response in a subject that issufficient to inhibit, neutralize or prevent infection of a broad rangeof influenza viruses (such as most or all influenza viruses within aspecific subtype). In some embodiments, the influenza protein isinfluenza HA or influenza NA. In one example, the optimized influenzaprotein is capable of eliciting a protective immune response againstmost or all known H5N1 influenza virus isolates (such as those listed inTable 1), such as about 80%, about 85%, about 90% or about 95% of knownH5N1 influenza virus isolates.

In some embodiments, the method of optimizing an influenza proteinsequence includes obtaining the amino acid sequences of a group ofinfluenza virus isolates. For example, the group can consist ofinfluenza virus isolates from a specific subtype (such as, for example,H5N1 or H1N1), and/or from one or more clades/sub-clades of a specificinfluenza subtype (for example, one or more of clades/sub-clades 0, 1,2.1, 2.2, 2.3, 2.4, 3, 4, 5, 6, 7, 8 and 9 of H5N1). Amino acidsequences of the group of influenza viruses are first organized by cladeor sub-clade and then by geographic location within each clade orsub-clade. The amino acid sequences for each geographic location arealigned to generate a primary consensus sequence for each geographicalregion. Grouping virus isolates by geographical region controls forsingle outbreak dominance and incomplete reporting and sequencing. Theprimary consensus sequence can be generated, for example, by multiplealignment analysis using AlignX (Vector NTI), or by any other methodknown in the art. The primary geographically-based consensus sequencesfor each clade or sub-clade are then aligned, and a secondary consensussequence is generated for each clade or sub-clade. The secondaryconsensus sequences for each clade or sub-clade are then aligned togenerate the optimized, broadly reactive, consensus sequence (see FIG.1). In some embodiments, the optimized influenza virus polypeptidesequence is further optimized for expression in mammalian cells. In someexamples, optimization includes reverse translation of the optimizedinfluenza virus polypeptide sequence to generate a coding sequence,followed by codon-optimization and/or optimization of the RNA (such asfor stability).

In one non-limiting example, the method of optimization includes: (i)obtaining the amino acid sequences of the polypeptide from a group ofinfluenza virus isolates, wherein the influenza virus isolates are fromthe same subtype; (ii) organizing the amino acid sequences of thepolypeptide from the group of influenza virus isolates by clade orsub-clade and then by geographical region within each clade orsub-clade; (iii) aligning the amino acid sequences within eachgeographical region to generate primary consensus sequences, whereineach geographic region is represented by a primary consensus sequence;(iv) aligning the primary consensus sequences to generate secondaryconsensus sequences, wherein each clade or sub-clade is represented by asecondary consensus sequence; and (v) aligning the secondary consensussequences to generate the optimized influenza virus polypeptidesequence. In some cases, the method further includes (i) reversetranslating the optimized influenza virus polypeptide sequence togenerate a coding sequence; and (ii) optimizing the coding sequence forexpression in mammalian cells.

In an alternative embodiment, the primary consensus sequence is obtainedby aligning influenza protein sequences (such as HA or NA sequences)from viral isolates from a single outbreak (a collection of influenzavirus isolates within a single country within a given year). Thus, inone non-limiting example, the method of optimization includes: (i)obtaining the amino acid sequences of the polypeptide from a group ofinfluenza virus isolates, wherein the influenza virus isolates are fromthe same subtype; (ii) organizing the amino acid sequences of thepolypeptide from the group of influenza virus isolates by clade orsub-clade and then by outbreak; (iii) aligning the amino acid sequenceswithin each outbreak to generate primary consensus sequences, whereineach outbreak is represented by a primary consensus sequence; (iv)aligning the primary consensus sequences to generate secondary consensussequences, wherein each clade or sub-clade is represented by a secondaryconsensus sequence; and (v) aligning the secondary consensus sequencesto generate the optimized influenza virus polypeptide sequence. In somecases, the method further includes (i) reverse translating the optimizedinfluenza virus polypeptide sequence to generate a coding sequence; and(ii) optimizing the coding sequence for expression in mammalian cells.

VI. Influenza

Influenza viruses are segmented negative-strand RNA viruses that belongto the Orthomyxoviridae family. There are three types of Influenzaviruses, A, B and C. Influenza A viruses infect a wide variety of birdsand mammals, including humans, horses, marine mammals, pigs, ferrets,and chickens. In animals, most influenza A viruses cause mild localizedinfections of the respiratory and intestinal tract. However, highlypathogenic influenza A strains, such as H5N1, cause systemic infectionsin poultry in which mortality may reach 100%. Animals infected withinfluenza A often act as a reservoir for the influenza viruses andcertain subtypes have been shown to cross the species barrier to humans.

Influenza A viruses can be classified into subtypes based on allelicvariations in antigenic regions of two genes that encode surfaceglycoproteins, namely, hemagglutinin (HA) and neuraminidase (NA) whichare required for viral attachment and cellular release. Currently,sixteen subtypes of HA (H1-H16) and nine NA (N-1-N9) antigenic variantsare known for influenza A virus. Previously, only three subtypes wereknown to circulate in humans (H1N1, H1N2, and H3N2). However, in recentyears, the pathogenic H5N1 subtype of avian influenza A has beenreported to cross the species barrier and infect humans as documented inHong Kong in 1997 and 2003, leading to the death of several patients.

In humans, the avian influenza virus infects cells of the respiratorytract as well as the intestinal tract, liver, spleen, kidneys and otherorgans. Symptoms of avian influenza infection include fever, respiratorydifficulties including shortness of breath and cough, lymphopenia,diarrhea and difficulties regulating blood sugar levels. In contrast toseasonal influenza, the group most at risk is healthy adults which makeup the bulk of the population. Due to the high pathogenicity of certainavian influenza A subtypes, particularly H5N1, and their demonstratedability to cross over to infect humans, there is a significant economicand public health risk associated with these viral strains, including areal epidemic and pandemic threat. Currently, no effective vaccines forH5N1 infection are available.

The influenza A virus genome encodes nine structural proteins and onenonstructural (NS1) protein with regulatory functions. The influenzavirus segmented genome contains eight negative-sense RNA (nsRNA) genesegments (PB2, PB1, PA, NP, M, NS, HA and NA) that encode at least tenpolypeptides, including RNA-directed RNA polymerase proteins (PB2, PB 1and PA), nucleoprotein (NP), neuraminidase (NA), hemagglutinin (subunitsHA1 and HA2), the matrix proteins (M1 and M2) and the non-structuralproteins (NS1 and NS2) (Krug et al., In “The Influenza Viruses,” R. M.Krug, ed., Plenum Press, N.Y., 1989, pp. 89 152).

Influenza virus' ability to cause widespread disease is due to itsability to evade the immune system by undergoing antigenic change, whichis believed to occur when a host is infected simultaneously with both ananimal influenza virus and a human influenza virus. During mutation andreassortment in the host, the virus may incorporate an HA and/or NAsurface protein gene from another virus into its genome, therebyproducing a new influenza subtype and evading the immune system.

HA is a viral surface glycoprotein generally comprising approximately560 amino acids and representing 25% of the total virus protein. It isresponsible for adhesion of the viral particle to, and its penetrationinto, a host cell in the early stages of infection. Cleavage of thevirus HA0 precursor into the HA1 and HA2 sub-fragments is a necessarystep in order for the virus to infect a cell. Thus, cleavage is requiredin order to convert new virus particles in a host cell into virionscapable of infecting new cells. Cleavage is known to occur duringtransport of the integral HA0 membrane protein from the endoplasmicreticulum of the infected cell to the plasma membrane. In the course oftransport, hemagglutinin undergoes a series of co- andpost-translational modifications including proteolytic cleavage of theprecursor HA into the amino-terminal fragment HAI and the carboxyterminal HA2. One of the primary difficulties in growing influenzastrains in primary tissue culture or established cell lines arises fromthe requirement for proteolytic cleavage activation of the influenzahemagglutinin in the host cell.

Although it is known that an uncleaved HA can mediate attachment of thevirus to its neuraminic acid-containing receptors on a cell surface, itis not capable of the next step in the infectious cycle, which isfusion. It has been reported that exposure of the hydrophobic aminoterminus of HA2 by cleavage is required so that it can be inserted intothe target cell, thereby forming a bridge between virus and target cellmembrane. This process is followed by fusion of the two membranes andentry of the virus into the target cell.

Proteolytic activation of HA involves cleavage at an arginine residue bya trypsin-like endoprotease, which is often an intracellular enzyme thatis calcium dependent and has a neutral pH optimum. Since the activatingproteases are cellular enzymes, the infected cell type determineswhether the HA is cleaved. The HA of the mammalian influenza viruses andthe nonpathogenic avian influenza viruses are susceptible to proteolyticcleavage only in a restricted number of cell types. On the other hand,HA of pathogenic avian viruses among the H5 and H7 subtypes are cleavedby proteases present in a broad range of different host cells. Thus,there are differences in host range resulting from differences inhemagglutinin cleavability which are correlated with the pathogenicproperties of the virus.

Neuraminidase (NA) is a second membrane glycoprotein of the influenzaviruses. The presence of viral NA has been shown to be important forgenerating a multi-faceted protective immune response against aninfecting virus. For most influenza A viruses, NA is 413 amino acid inlength, and is encoded by a gene of 1413 nucleotides. Nine different NAsubtypes have been identified in influenza viruses (N1, N2, N3, N4, N5,N6, N7, N8 and N9), all of which have been found among wild birds. NA isinvolved in the destruction of the cellular receptor for the viral HA bycleaving terminal neuraminic acid (also called sialic acid) residuesfrom carbohydrate moieties on the surfaces of infected cells. NA alsocleaves sialic acid residues from viral proteins, preventing aggregationof viruses. Using this mechanism, it is hypothesized that NA facilitatesrelease of viral progeny by preventing newly formed viral particles fromaccumulating along the cell membrane, as well as by promotingtransportation of the virus through the mucus present on the mucosalsurface. NA is an important antigenic determinant that is subject toantigenic variation.

In addition to the surface proteins HA and NA, influenza virus comprisessix additional internal genes, which give rise to eight differentproteins, including polymerase genes PB1, PB2 and PA, matrix proteins M1and M2, nucleoprotein (NP), and non-structural proteins NS1 and NS2(Horimoto et al., Clin Microbiol Rev. 14(1):129-149, 2001).

In order to be packaged into progeny virions, viral RNA is transportedfrom the nucleus as a ribonucleoprotein (RNP) complex composed of thethree influenza virus polymerase proteins, the nucleoprotein (NP), andthe viral RNA, in association with the influenza virus matrix 1 (M1)protein and nuclear export protein (Marsh et al., J. Virol,82:2295-2304, 2008). The M1 protein that lies within the envelope isthought to function in assembly and budding. A limited number of M2proteins are integrated into the virions (Zebedee, J. Virol.62:2762-2772, 1988). They form tetramers having H+ ion channel activity,and when activated by the low pH in endosomes, acidify the inside of thevirion, facilitating its uncoating (Pinto et al., Cell 69:517-528,1992). Amantadine is an anti-influenza drug that prevents viralinfection by interfering with M2 ion channel activity, thus inhibitingvirus uncoating.

NS1, a nonstructural protein, has multiple functions, includingregulation of splicing and nuclear export of cellular mRNAs as well asstimulation of translation. The major function of NS1 seems to be tocounteract the interferon activity of the host, since an NS1 knockoutvirus was viable although it grew less efficiently than the parent virusin interferon-nondefective cells (Garcia-Sastre, Virology 252:324-330,1998).

NS2 has been detected in virus particles (Richardson et al., Arch.Virol. 116:69-80, 1991; Yasuda et al., Virology 196:249-255, 1993). Theaverage number of NS2 proteins in a virus particle was estimated to be130-200 molecules. An in vitro binding assay shows directprotein-protein contact between M1 and NS2. NS2-M1 complexes have alsobeen detected by immunoprecipitation in virus-infected cell lysates. TheNS2 protein is thought to play a role in the export of RNP from thenucleus through interaction with M1 protein (Ward et al., Arch. Virol.140:2067-2073, 1995).

V. Influenza Proteins, VLPs and Administration Thereof

Optimized influenza HA polypeptides and influenza VLPs comprising anoptimized HA (such as the HA having the sequence set forth as SEQ ID NO:2) are provided herein. The optimized HA polypeptides can beadministered to elicit an immune response against influenza. Inparticular examples, the optimized HA polypeptides are administered aspart of a VLP.

The influenza VLPs are made up of the HA, NA and M1 proteins. Theproduction of influenza VLPs has been described in the art and is withinthe abilities of one of ordinary skill in the art. As described herein,influenza VLPs can be produced by transfection of host cells withplasmids encoding the HA, NA and M1 proteins. After incubation of thetransfected cells for an appropriate time to allow for proteinexpression (such as for approximately 72 hours), VLPs can be isolatedfrom cell culture supernatants. Example 1 below provides an exemplaryprotocol for purifying influenza VLPs from cell supernatants. In thisexample, VLPs are isolated by low speed centrifugation (to remove celldebris), vacuum filtration and ultracentrifugation through 20% glycerol.

The influenza VLPs disclosed herein can be used as influenza vaccines toelicit a protective immune response against H5N1 influenza viruses.

Influenza HA polypeptides and VLPs comprising HA polypeptides, orcompositions thereof, can be administered to a subject by any of theroutes normally used for introducing recombinant virus into a subject.Methods of administration include, but are not limited to, intradermal,intramuscular, intraperitoneal, parenteral, intravenous, subcutaneous,vaginal, rectal, intranasal, inhalation or oral. Parenteraladministration, such as subcutaneous, intravenous or intramuscularadministration, is generally achieved by injection. Injectables can beprepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution or suspension in liquidprior to injection, or as emulsions. Injection solutions and suspensionscan be prepared from sterile powders, granules, and tablets of the kindpreviously described. Administration can be systemic or local.

Influenza VLPs, or compositions thereof, are administered in anysuitable manner, such as with pharmaceutically acceptable carriers.Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent disclosure.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

Administration can be accomplished by single or multiple doses. The doseadministered to a subject in the context of the present disclosureshould be sufficient to induce a beneficial therapeutic response in asubject over time, or to inhibit or prevent H5N1 influenza virusinfection. The dose required will vary from subject to subject dependingon the species, age, weight and general condition of the subject, theseverity of the infection being treated, the particular compositionbeing used and its mode of administration. An appropriate dose can bedetermined by one of ordinary skill in the art using only routineexperimentation.

Provided herein are pharmaceutical compositions which include atherapeutically effective amount of the influenza VLPs alone or incombination with a pharmaceutically acceptable carrier. Pharmaceuticallyacceptable carriers include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol, and combinations thereof.The carrier and composition can be sterile, and the formulation suitsthe mode of administration. The composition can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thecomposition can be a liquid solution, suspension, emulsion, tablet,pill, capsule, sustained release formulation, or powder. The compositioncan be formulated as a suppository, with traditional binders andcarriers such as triglycerides. Oral formulations can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, and magnesiumcarbonate. Any of the common pharmaceutical carriers, such as sterilesaline solution or sesame oil, can be used. The medium can also containconventional pharmaceutical adjunct materials such as, for example,pharmaceutically acceptable salts to adjust the osmotic pressure,buffers, preservatives and the like. Other media that can be used withthe compositions and methods provided herein are normal saline andsesame oil.

The influenza VLPs described herein can be administered alone or incombination with other therapeutic agents to enhance antigenicity. Forexample, the influenza VLPs can be administered with an adjuvant, suchas Freund incomplete adjuvant or Freund's complete adjuvant.

Optionally, one or more cytokines, such as IL-2, IL-6, IL-12, RANTES,GM-CSF, TNF-α, or IFN-γ, one or more growth factors, such as GM-CSF orG-CSF; one or more molecules such as OX-40L or 41 BBL, or combinationsof these molecules, can be used as biological adjuvants (see, forexample, Salgaller et al., 1998, J. Surg. Oncol. 68(2):122-38; Lotze etal., 2000, Cancer J. Sci. Am. 6(Suppl 1):S61-6; Cao et al., 1998, StemCells 16(Suppl 1):251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol.465:381-90). These molecules can be administered systemically (orlocally) to the host.

A number of means for inducing cellular responses, both in vitro and invivo, are known. Lipids have been identified as agents capable ofassisting in priming CTL in vivo against various antigens. For example,as described in U.S. Pat. No. 5,662,907, palmitic acid residues can beattached to the alpha and epsilon amino groups of a lysine residue andthen linked (for example, via one or more linking residues, such asglycine, glycine-glycine, serine, serine-serine, or the like) to animmunogenic peptide. The lipidated peptide can then be injected directlyin a micellar form, incorporated in a liposome, or emulsified in anadjuvant. As another example, E. coli lipoproteins, such astripalmitoyl-S-glycerylcysteinlyseryl-serine can be used to prime tumorspecific CTL when covalently attached to an appropriate peptide (see,Deres et al., Nature 342:561. 1989). Further, as the induction ofneutralizing antibodies can also be primed with the same moleculeconjugated to a peptide which displays an appropriate epitope, twocompositions can be combined to elicit both humoral and cell-mediatedresponses where that is deemed desirable.

Although administration of VLPs containing the optimized HA protein, oneof skill in the art would understand that it is also possible toadminister the optimized influenza HA protein itself (in the absence ofa viral particle) or as a fusion protein to elicit an immune response ina subject.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLES Example 1 A Computationally Optimized Broadly Reactive Antigen(COBRA) Based H5N1 VLP Vaccine Elicits Broadly Reactive Antibodies inMice and Ferrets

This example describes the finding that mice and ferrets vaccinated withCOBRA hemagglutinin (HA) H5N1 VLPs exhibited protective levels of HAIantibodies to representative isolates from each sub-clade of clade 2 andwere completely protected from lethal challenge with a clade 2.2 H5N1virus.

Materials and Methods COBRA Hemagglutinin (HA) Construction andSynthesis

Influenza A HA amino acid sequences isolated from human H5N1 infectionswere downloaded from the NCBI Influenza Virus Resource database (Bao etal., J Virol 82:596-601, 2008; see Table 1 for a complete list ofaccession numbers and isolate descriptions). Nucleotide sequences weretranslated into protein sequences using the standard genetic code. Allavailable full length sequences from H5N1 clade 2 human infections from2004 to 2006 were acquired and used for subsequent consensusgenerations. For each round of consensus generation, multiple alignmentanalysis was applied and the consensus sequence was generated usingAlignX (Vector NTI). The final amino acid sequence, termedcomputationally optimized broadly reactive antigen (COBRA), was reversetranslated and optimized for expression in mammalian cells, includingcodon usage and RNA optimization (GeneArt; Regensburg, Germany). Thisconstruct was then synthesized and inserted into the pTR600 expressionvector (U.S. Patent Application Publication No. 2002/0106798; Ross etal., Nat. Immunol. 1(2):102-103, 2000; Green et al., Vaccine 20:242-248,2001).

TABLE 1 COBRA input sequences Strain Clade Accession Host Country YearA/Indonesia/534H/2006 2.1.2 EU146737 Human Indonesia 2006A/Indonesia/535H/2006 2.1.2 EU146753 Human Indonesia 2006A/Indonesia/536H/2006 2.1.2 EU146754 Human Indonesia 2006A/Indonesia/538H/2006 2.1.2 EU146745 Human Indonesia 2006A/Indonesia/546bH/2006 2.1.2 EU146793 Human Indonesia 2006A/Indonesia/546H/2006 2.1.2 EU146755 Human Indonesia 2006A/Indonesia/560H/2006 2.1.2 EU146785 Human Indonesia 2006A/Indonesia/CDC594/2006 2.1.2 CY014272 Human Indonesia 2006A/Indonesia/CDC595/2006 2.1.2 CY014280 Human Indonesia 2006A/Indonesia/CDC596/2006 2.1.2 CY014288 Human Indonesia 2006A/Indonesia/CDC597/2006 2.1.2 CY014296 Human Indonesia 2006A/Indonesia/CDC599/2006 2.1.2 CY014303 Human Indonesia 2006A/Indonesia/CDC599N/2006 2.1.2 CY014477 Human Indonesia 2006A/Indonesia/CDC625/2006 2.1.2 CY014433 Human Indonesia 2006A/Indonesia/CDC625L/2006 2.1.2 CY014465 Human Indonesia 2006A/Indonesia/160H/2005 2.1.3 EU146648 Human Indonesia 2005A/Indonesia/175H/2005 2.1.3 EU146640 Human Indonesia 2005A/Indonesia/177H/2005 2.1.3 EU146680 Human Indonesia 2005A/Indonesia/195H/2005 2.1.3 EU146656 Human Indonesia 2005A/Indonesia/239H/2005 2.1.3 EU146664 Human Indonesia 2005A/Indonesia/245H/2005 2.1.3 EU146672 Human Indonesia 2005A/Indonesia/283H/2006 2.1.3 EU146681 Human Indonesia 2006A/Indonesia/286H/2006 2.1.3 EU146688 Human Indonesia 2006A/Indonesia/292H/2006 2.1.3 EU146713 Human Indonesia 2006A/Indonesia/298H/2006 2.1.3 EU146697 Human Indonesia 2006A/Indonesia/304H/2006 2.1.3 EU146705 Human Indonesia 2006A/Indonesia/321H/2006 2.1.3 EU146721 Human Indonesia 2006A/Indonesia/341H/2006 2.1.3 EU146729 Human Indonesia 2006A/Indonesia/5/2005 2.1.3 EF541394 Human Indonesia 2005A/Indonesia/542H/2006 2.1.3 EU146777 Human Indonesia 2006A/Indonesia/567H/2006 2.1.3 EU146801 Human Indonesia 2006A/Indonesia/569H/2006 2.1.3 EU146809 Human Indonesia 2006A/Indonesia/583H/2006 2.1.3 EU146817 Human Indonesia 2006A/Indonesia/604H/2006 2.1.3 EU146825 Human Indonesia 2006A/Indonesia/7/2005 2.1.3 EU146632 Human Indonesia 2005A/Indonesia/CDC184/2005 2.1.3 CY014197 Human Indonesia 2005A/Indonesia/CDC194P/2005 2.1.3 CY014168 Human Indonesia 2005A/Indonesia/CDC287E/2005 2.1.3 CY014198 Human Indonesia 2005A/Indonesia/CDC287T/2005 2.1.3 CY014199 Human Indonesia 2005A/Indonesia/CDC292N/2005 2.1.3 CY014200 Human Indonesia 2005A/Indonesia/CDC292T/2005 2.1.3 CY014201 Human Indonesia 2005A/Indonesia/CDC326/2006 2.1.3 CY014204 Human Indonesia 2006A/Indonesia/CDC326N/2006 2.1.3 CY014202 Human Indonesia 2006A/Indonesia/CDC326N2/2006 2.1.3 CY014203 Human Indonesia 2006A/Indonesia/CDC326T/2006 2.1.3 CY014205 Human Indonesia 2006A/Indonesia/CDC329/2006 2.1.3 CY014206 Human Indonesia 2006A/Indonesia/CDC357/2006 2.1.3 CY014207 Human Indonesia 2006A/Indonesia/CDC370/2006 2.1.3 CY014209 Human Indonesia 2006A/Indonesia/CDC370E/2006 2.1.3 CY014210 Human Indonesia 2006A/Indonesia/CDC370P/2006 2.1.3 CY014211 Human Indonesia 2006A/Indonesia/CDC370T/2006 2.1.3 CY014212 Human Indonesia 2006A/Indonesia/CDC390/2006 2.1.3 CY014213 Human Indonesia 2006A/Indonesia/CDC523/2006 2.1.3 CY014311 Human Indonesia 2006A/Indonesia/CDC523E/2006 2.1.3 CY014368 Human Indonesia 2006A/Indonesia/CDC523T/2006 2.1.3 CY014376 Human Indonesia 2006A/Indonesia/CDC582/2006 2.1.3 CY014384 Human Indonesia 2006A/Indonesia/CDC610/2006 2.1.3 CY014393 Human Indonesia 2006A/Indonesia/CDC623/2006 2.1.3 CY014401 Human Indonesia 2006A/Indonesia/CDC623E/2006 2.1.3 CY014409 Human Indonesia 2006A/Indonesia/CDC624/2006 2.1.3 CY014417 Human Indonesia 2006A/Indonesia/CDC624E/2006 2.1.3 CY014425 Human Indonesia 2006A/Indonesia/CDC634/2006 2.1.3 CY014441 Human Indonesia 2006A/Indonesia/CDC634P/2006 2.1.3 CY014449 Human Indonesia 2006A/Indonesia/CDC634T/2006 2.1.3 CY014457 Human Indonesia 2006A/Indonesia/CDC644/2006 2.1.3 CY014518 Human Indonesia 2006A/Indonesia/CDC644T/2006 2.1.3 CY014510 Human Indonesia 2006A/Indonesia/CDC669/2006 2.1.3 CY014481 Human Indonesia 2006A/Indonesia/CDC669P/2006 2.1.3 CY014489 Human Indonesia 2006A/Indonesia/CDC699/2006 2.1.3 CY014497 Human Indonesia 2006A/Indonesia/CDC7/2005 2.1.3 CY014177 Human Indonesia 2005A/Indonesia/CDC739/2006 2.1.3 CY014529 Human Indonesia 2006A/Indonesia/CDC759/2006 2.1.3 CY014543 Human Indonesia 2006A/Indonesia/CDC835/2006 2.1.3 CY017662 Human Indonesia 2006A/Indonesia/CDC836/2006 2.1.3 CY017670 Human Indonesia 2006A/Indonesia/CDC836T/2006 2.1.3 CY017678 Human Indonesia 2006A/Indonesia/CDC887/2006 2.1.3 CY017688 Human Indonesia 2006A/Indonesia/CDC938/2006 2.1.3 CY017638 Human Indonesia 2006A/Indonesia/CDC938E/2006 2.1.3 CY017646 Human Indonesia 2006A/Indonesia/CDC940/2006 2.1.3 CY017654 Human Indonesia 2006A/Indonesia/TLL001/2006 2.1.3 EU015403 Human Indonesia 2006A/Indonesia/TLL002/2006 2.1.3 EU015404 Human Indonesia 2006A/Indonesia/TLL003/2006 2.1.3 EU015405 Human Indonesia 2006A/Indonesia/TLL004/2006 2.1.3 EU015406 Human Indonesia 2006A/Indonesia/TLL005/2006 2.1.3 EU015407 Human Indonesia 2006A/Indonesia/TLL006/2006 2.1.3 EU015408 Human Indonesia 2006A/Indonesia/TLL007/2006 2.1.3 EU015409 Human Indonesia 2006A/Indonesia/TLL008/2006 2.1.3 EU015410 Human Indonesia 2006A/Indonesia/TLL009/2006 2.1.3 EU015411 Human Indonesia 2006A/Indonesia/TLL010/2006 2.1.3 EU015412 Human Indonesia 2006A/Indonesia/TLL011/2006 2.1.3 EU015413 Human Indonesia 2006A/Indonesia/TLL012/2006 2.1.3 EU015414 Human Indonesia 2006A/Indonesia/TLL013/2006 2.1.3 EU015415 Human Indonesia 2006A/Indonesia/TLL014/2006 2.1.3 EU015416 Human Indonesia 2006A/Djibouti/5691NAMRU3/2006 2.2 DQ666146 Human Djibouti 2006A/Egypt/7021-NAMRU3/2006 2.2 CY062439 Human Egypt 2006A/human/Iraq/207-NAMRU3/2006 2.2 DQ435202 Human Iraq 2006 A/Iraq/1/20062.2 EU146870 Human Iraq 2006 A/Iraq/659/2006 2.2 EU146876 Human Iraq2006 A/Iraq/754/2006 2.2 EU146877 Human Iraq 2006 A/Iraq/755/2006 2.2EU146869 Human Iraq 2006 A/Iraq/756/2006 2.2 EU146878 Human Iraq 2006A/Turkey/12/2006 2.2 EF619982 Human Turkey 2006 A/Turkey/15/2006 2.2EF619989 Human Turkey 2006 A/Turkey/651242/2006 2.2 EF619990 HumanTurkey 2006 A/Turkey/65596/2006 2.2 EF619998 Human Turkey 2006A/Xinjiang/1/2006 2.2 FJ492886 Human China 2006A/Egypt/14724-NAMRU3/2006 2.2.1 EF200512 Human Egypt 2006A/Egypt/14725-NAMRU3/2006 2.2.1 EF200513 Human Egypt 2006A/Egypt/2782-NAMRU3/2006 2.2.1 DQ464377 Human Egypt 2006A/Egypt/2991-NAMRU3/2006 2.2.1 EU095023 Human Egypt 2006A/Egypt/2992-NAMRU3/2006 2.2.1 EU095024 Human Egypt 2006A/Egypt/902782/2006 2.2.1 EU146867 Human Egypt 2006 A/Egypt/902786/20062.2.1 EU146868 Human Egypt 2006 A/Anhui/1/2005 2.3.4 DQ371928 HumanChina 2005 A/Anhui/2/2005 2.3.4 DQ371929 Human China 2005 A/China/20062.3.4 EF624256 Human China 2006 A/China/GD01/2006 2.3.4 DQ835313 HumanChina 2006 A/Fujian/1/2005 2.3.4 FJ492882 Human China 2005A/Guangdong/1/2006 2.3.4 FJ492884 Human China 2006 A/Guangxi/1/20052.3.4 DQ371930 Human China 2005 A/human/China/GD02/2006 2.3.4 EU263981Human China 2006 A/Hunan/1/2006 2.3.4 FJ492879 Human China 2006A/Jiangxi/1/2005 2.3.4 FJ492885 Human China 2005 A/Shanghai/1/2006 2.3.4AB462295 Human China 2006 A/Shenzhen/406H/2006 2.3.4 EF137706 HumanChina 2006 A/Sichuan/1/2006 2.3.4 FJ492881 Human China 2006A/Vietnam/UT30850/2005 2.3.4 HM114537 Human Viet Nam 2005A/Zhejiang/1/2006 2.3.4 FJ492880 Human China 2006 A/Zhejiang/16/20062.3.4 DQ643809 Human China 2006

COBRA HA Antigenic Modeling

Influenza hemagglutinin (HA) protein sequences representing COBRA,A/Indonesia/5/2005 (Clade 2.1), A/Whooper Swan/Mongolia/244/2005 (Clade2.2) and A/Anhui/1/2005 (Clade 2.3) were submitted to the 3D-JIGSAWProtein Comparative Modeling website for rendering (Bates et al.,Proteins 45(S5):39-46, 2001; Bates and Sternberg, Proteins 37(53):47-54,1999; Contreras-Moreira and Bates, Bioinformatics 18(8):1141-1142,2002). Structures were overlaid and analyzed using Swiss-Pdb viewersoftware (Guex and Peitsch, Electrophoresis 18(15):2714-23, 1998).Antigenic regions and designations are based on the original descriptionof the antigenic structure of the H3N2 virus A/Hong Kong/1/1968 (Wileyet al., Nature 289(5796):373-378, 1981). No significant alterations wereobserved in region B of the COBRA HA relative to the primary influenzaisolates; however, some divergent structures in HA regions A and C wereidentified in primary isolates.

In Vitro Expression

COBRA HA protein expression was confirmed by transfecting mammaliancells. Human embryonic kidney (HEK) 293T cells (1×10⁶) were transientlytransfected with 3 μg of DNA expressing COBRA. Cells were incubated for72 hours at 37° C., supernatants were removed, the cells were lysed with1% Triton-X 100 and cell lysates were collected. Cell lysates wereelectrophoresed on a 10% SDS-PAGE gel and transferred to a PVDFmembrane. The blot was probed with mouse polyclonal antisera pooled frommice infected with 6:2 reassortant H5N1 viruses with the surfaceglycoproteins derived from either A/Vietnam/1203/2004 or A/WhooperSwan/244/2005, and the HA-antibody complexes were detected using a goatanti-mouse IgG conjugated to horse radish peroxidase (HRP) (SouthernBiotech; Birmingham, Ala., USA). HRP was detected by chemiluminescentsubstrate (Pierce Biotechnology; Rockford Ill., USA) and exposed toX-ray film (ThermoFisher; Pittsburgh, Pa., USA).

COBRA HA Functional Characterization

To determine receptor binding characteristics, virus-like particles(VLPs) containing COBRA HA proteins were purified from the supernatantsof mammalian cell lines. HEK 293T cells were transiently transfectedwith plasmids expressing HIV Gag, COBRA HA and neuraminidase (NA,A/Thailand/1(KAN-1)/2004) and incubated for 72 hours at 37° C.Supernatants were collected and VLPs were purified viaultracentrifugation (100,000×g through 20% glycerol, weight per volume)for 4 hours at 4° C. The pellets were subsequently resuspended inphosphate buffered saline PBS, pH 7.2 and stored at −80° C. until use.Protein concentration was determined by Micro BCA™ Protein Assay ReagentKit (Pierce Biotechnology, Rockford, Ill., USA). COBRA HA VLPs wereprepared in various amounts as measured by total protein and eachindividual preparation was two-fold serially diluted in v-bottommicrotiter plates. An equal volume of either 1% turkey or 1% horseerythrocytes (RBC) (Lampire; Pipersville, Pa., USA) in PBS was added tothe diluted VLPs and incubated for 30-60 minutes at room temperature.The HA titer was determined by the reciprocal dilution of the last wellwhich contained agglutinated RBC.

To determine endosomal fusion characteristics. COBRA-pseudotypedlentiviral vectors expressing a luciferase reporter gene were producedas described (Yang et al., J Virol 78(8):4029-4036). Briefly, 293T cellswere co-transfected by using the following plasmids: 7 μg ofpCMVdeltaR8.2, 7 μg of pHRCMV-Luc, 3 μg pCMV/R N1(Kan-1) and 3 μg pTR600COBRA. Cells were transiently transfected and incubated for 72 hours at37° C. Supernatants were harvested, centrifuged to clear cell debris,filtered through a 0.22 μm syringe filter, aliquotted, and usedimmediately or frozen at −80° C. For fusion assays, 100 μl ofpseudoviruses were added to confluent MDCK cells in 48-well plates(˜30,000 cells per well). Plates were incubated at room temperature for30 minutes, washed, and fresh medium added. Forty-eight hours afterinfection, cells were lysed in mammalian cell lysis buffer (Promega;Madison, Wis., USA). A standard quantity of cell lysate was used in aluciferase assay with luciferase assay reagent (Promega; Madison, Wis.,USA) according to the manufacturer's protocol.

Vaccine Preparation and Dose Determination

HEK 293T cells were transiently transfected with plasmids expressing M1(A/Puerto Rico/8/1934, optimized for expression in mammalian cells; SEQID NO: 9), NA (A/Thailand/1(KAN-1)/2004, optimized for expression inmammalian cells; SEQ ID NO: 8) and COBRA HA (SEQ ID NO: 7) and incubatedfor 72 hours at 37° C. Supernatants were collected and cell debrisremoved by low speed centrifugation followed by vacuum filtrationthrough a 0.22 μm sterile filter. VLPs were purified viaultracentrifugation (100,000×g through 20% glycerol, weight per volume)for 4 hours at 4° C. The pellets were subsequently resuspended in PBS pH7.2 and stored in single use aliquots at −80° C. until use. Totalprotein concentration was determined by Micro BCA™ Protein Assay ReagentKit (Pierce Biotechnology, Rockford, Ill., USA).

HA specific content was determined by western blot and densitometry.Purified recombinant COBRA HA and purified VLPs were prepared instandard total protein amounts and were electrophoresed on a 10%SDS-PAGE gel and transferred to a PVDF membrane. The blot was probedwith mouse polyclonal antisera pooled from mice infected with 6:2reassortant H5N1 viruses with the surface glycoproteins derived fromeither A/Vietnam/1203/2004 or A/Whooper Swan/244/2005 and theHA-antibody complexes were detected using a goat anti-mouse IgGconjugated to horse radish peroxidase (HRP) (Southern Biotech;Birmingham, Ala., USA). HRP was detected by chemiluminescent substrate(Pierce Biotechnology; Rockford Ill., USA) and exposed to X-ray film(ThermoFisher; Pittsburgh, Pa., USA). Density of bands was determinedusing ImageJ software (NIH) (Abramoff et al., Biophotonics International11(7):36-42, 2004). Density of recombinant HA bands were used tocalculate a standard curve and the density of the purified VLPs wasinterpolated using the results from the recombinant HA. Experiments wereperformed in triplicate and multiple exposure times were analyzed forall iterations.

Codon-Optimized Influenza HA, NA and M1 Genes

The nucleotide sequences of the codon-optimized HA (SEQ ID NO: 1),codon-optimized NA (SEQ ID NO: 3) and codon-optimized M1 (SEQ ID NO: 5)genes are set forth in the Sequence Listing. The corresponding aminoacid sequences of the encoded HA, NA and M1 proteins are set forth inthe Sequence Listing as SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6,respectively.

Mouse Studies

BALB/c mice (Mus musculis, females, 6-8 weeks old) were purchased fromHarlan Sprague Dawley (Indianapolis, Ind., USA) and housed inmicroisolator units and allowed free access to food and water and werecared for under USDA guidelines for laboratory animals. Mice (5 mice pergroup) were vaccinated with one of three doses of purified COBRA HA VLPs(1.5 μg, 0.3 μg or 0.06 μg), based upon HA content from a densitometryassay, via intramuscular injection at week 0 and then boosted with thesame dose at week 3. For comparison studies, mice (20 mice per group)were vaccinated with purified VLPs (3 μg) via intramuscular injection atweek 0 and then boosted with the same dose at week 3. Vaccines at eachdose were formulated with Imject® alum adjuvant (Imject® Alum, PierceBiotechnology; Rockford, Ill., USA) according to the manufacturer'sprotocol or vehicle alone. Fourteen to twenty-one days after eachvaccination, blood was collected from anesthetized mice via theretro-orbital plexus and transferred to a microfuge tube. Tubes werecentrifuged and sera was removed and frozen at −20±5° C.

Three weeks after final vaccination, mice were challenged intranasallywith 5×10³ plaque forming units (PFU) of the highly pathogenic H5N1virus A/Whooper Swan/Mongolia/244/2005 (clade 2.2) in a volume of 50 μl.The challenge dose represents approximately 50 LD₅₀ in mice. Afterinfection, mice were monitored daily for weight loss, disease signs anddeath for 14 days after infection. Individual body weights, sicknessscores and death were recorded for each group on each day afterinoculation. Sickness score was determined by evaluating activity(0=normal, 1=reduced, 2=severely reduced), hunched back (0=absent,1=present) and ruffled fur (0=absent, 1=present) (Toapanta and Ross,Respiratory Res 10(1):112, 2009). Experimental endpoint was definedas >20% weight loss or display of neurological disease such as hind limbparalysis. All H5N1 influenza virus studies were performed underhigh-containment biosafety level 3 enhanced conditions (BSL3+).

Ferret Studies

Fitch ferrets (Mustela putorius faro, female, 6-12-months of age),influenza naïve and de-scented, were purchased from Marshall Farms(Sayre, Pa., USA). Ferrets were pair housed in stainless steel cages(Shor-line, Kansas City, Kans., USA) containing Sani-chips LaboratoryAnimal Bedding (P.J. Murphy Forest Products, Montville, N.J., USA).Ferrets were provided with Teklad Global Ferret Diet (Harlan Teklad,Madison, Wis., USA) and fresh water ad libitum. The COBRA HA VLPs werediluted in PBS, pH 7.2 to achieve final concentration. Ferrets (n=3)were vaccinated with 15 kg of purified COBRA VLPs, based upon HA contentas determined by densitometry assay, via intramuscular injection in thequadriceps muscle in a volume of 0.25 ml at week 0 and then boosted withthe same dose at week 3. Vaccines were stored at −80° C. prior to useand formulated with Imject® alum adjuvant (Imject® Alum; PierceBiotechnology, Rockford, Ill., USA) immediately prior to use. Animalswere monitored for adverse events including weight loss, temperature,loss of activity, nasal discharge, sneezing and diarrhea weekly duringthe vaccination regimen. Prior to vaccination, animals were confirmed byHAI assay to be seronegative for circulating influenza A (H1N1 and H3N2)and influenza B viruses. Fourteen to twenty-one days after eachvaccination, blood was collected from anesthetized ferrets via theanterior vena cava and transferred to a microfuge tube. Tubes werecentrifuged and sera was removed and frozen at −20±5° C.

Three weeks after final vaccination, ferrets were challengedintranasally with 1×10⁶ plaque forming units (PFU) of the highlypathogenic H5N1 virus A/Whooper Swan/Mongolia/244/2005 (clade 2.2) in avolume of 0.5 ml in each nostril for a total infection volume of 1 ml.After infection, ferrets were monitored daily for weight loss, diseasesigns and death for 14 days after infection. Individual body weights,sickness scores, and death were recorded for each group on each dayafter inoculation. Sickness score was determined by evaluating activity(0=normal, 1=alert and active with stimulation, 2=alert but not activeafter stimulation, 3=not alert or active after stimulation), nasaldischarge (0=absent, 1=present), sneezing (0=absent, 1=present),decreased food intake (0=absent, 1=present), diarrhea (0=absent,1=present), dyspnea (0=absent, 1=present) and neurological symptoms(0=absent, 1=present). Nasal washes were performed by instilling 3 ml ofPBS into the nares of anesthetized ferrets each day for 14 days afterinoculation. Washes were collected and stored at −80° C. until use.Experimental endpoint was defined as >20% weight loss, development ofneurological symptoms, or an activity score of 3 (not active or alertafter stimulation). All H5N1 influenza virus studies were performedunder high-containment biosafety level 3 enhanced conditions (BSL3+).

ELISA

The ELISA assay was used to assess total antibody titer and IgG isotypetiter to the HA. High binding, 96-well polystyrene plates (Costar;Lowell, Mass., USA) were coated overnight with 50 ng/well of recombinantHA. Coating antigens were derived from the following representativeviral isolates: A/Vietnam/1203/2004 (clade 1), A/Indonesia/5/2005 (clade2.1), A/Whooper Swan/244/2005 (clade 2.2) and A/Anhui/1/2005 (clade2.3). Plates were blocked with 5% milk diluted in PBS with 0.05% Tween20. Serum samples were diluted in blocking buffer and added to plates.Serum was two-fold serially diluted and allowed to incubate for 1 hourat room temperature. Plates were washed and species specific antibodyagainst IgG, IgG1, IgG2a, IgG2b or IgG3 and linked to horseradishperoxidase (HRP) (Southern Biotech; Birmingham, Ala., USA) were dilutedin blocking buffer and added to plates. Plates were incubated for 1 hourat room temperature. Plates were washed and HRP was developed with TMBsubstrate (Sigma-Aldrich; St. Louis, Mo., USA). Plates were incubated inthe dark for 15 minutes and then the reaction was stopped with 2N H₂SO₄.Optical densities at a wavelength of 450 nm (OD₄₅₀) were read by aspectrophotometer (BioTek; Winooski, Vt., USA) and end point dilutiontiters were determined. End point titers were determined as thereciprocal dilution of the last well which had an OD₄₅₀ above the meanOD₄₅₀ plus two standard deviations of naïve animal sera.

Hemagglutination Inhibition (HAI)

The HAI assay was used to assess functional antibodies to HA able toinhibit agglutination of horse erythrocytes. The protocol was adaptedfrom the CDC laboratory-based influenza surveillance manual (Gillim-Rossand Subbarao, Clin Microbiol Rev 19(4):614-636, 2006). To inactivatenon-specific inhibitors, sera were treated with receptor destroyingenzyme (RDE; Denka Seiken, Co., Japan) prior to being tested (Bright etal. Lancet 366(9492):1175-1181, 2005; Bright et al., Virology308(2):270-278, 2003; Bright et al., JAMA 295(8):891-894, 2006; Mitchellet al., Vaccine 21(9-10):902-914, 2004; Ross et al., Nat Immunol1(2):127-131, 2000). Briefly, three parts RDE was added to one part seraand incubated overnight at 37° C. RDE was inactivated by incubation at56° C. for ˜30 minutes. RDE-treated sera was two-fold serially dilutedin v-bottom microtiter plates. An equal volume of reassortant virus,adjusted to approximately 8 HAU/50 was added to each well. Thereassortant viruses contained the internal genes from the mouse adaptedstrain A/Puerto Rico/8/1934 and the surface proteins HA and NA from thefollowing representative viral isolates: A/Vietnam/1203/2004 (clade 1),A/Indonesia/5/2005 (clade 2.1), A/Whooper Swan/244/2005 (clade 2.2) andA/Anhui/1/2005 (clade 2.3). The plates were covered and incubated atroom temperature for 20 minutes followed by the addition of 1% horseerythrocytes (hRBC) (Lampire Biologicals, Pipersville, Pa., USA) in PBS.Red blood cells were stored at 4° C. and used within 72 hours ofpreparation. The plates were mixed by agitation, covered, and the RBCswere allowed to settle for 1 hour at room temperature (Askonas B,McMichael A, Webster R. The immune response to influenza viruses and theproblem of protection against infection. In: Beare A S, editor. Basicand applied influenza research: CRC Press 1982: 159-188). The HAI titerwas determined by the reciprocal dilution of the last row whichcontained non-agglutinated RBCs. Positive and negative serum controlswere included for each plate. All mice were negative (HAI≦1:10) forpre-existing antibodies to currently circulating human influenza virusesprior to vaccination.

Plaque Assay

Madin-Darby Canine Kidney (MDCK) cells were plated (5×10⁵) in each wellof a 6-well plate. Samples were diluted (final dilution factors of 10°to 10⁻⁶) and overlayed onto the cells in 100 μl of DMEM supplementedwith penicillin-streptomycin and incubated for 1 hour. Samples wereremoved, cells were washed twice and media was replaced with 2 ml of L15medium plus 0.8% agarose (Cambrex; East Rutherford, N.J., USA) andincubated for 72 hours at 37° C. with 5% CO₂. Agarose was removed anddiscarded. Cells were fixed with 10% buffered formalin, and then stainedwith 1% crystal violet for 15 minutes. Following thorough washing indH₂O to remove excess crystal violet, plates were allowed to dry,plaques counted, and the plaque forming units (PFU)/ml were calculated.

Statistical Analysis

Statistical significance of the antibody data was determined using atwo-way analysis of variance (ANOVA) with Bonferroni's post-test toanalyze differences between each vaccine group for the different testantigens (multiparametric). Differences in weight loss, sickness score,and viral titers were analyzed by two-way ANOVA, followed byBonferroni's post test for each vaccine group at multiple time points.Significance was defined as p<0.05. Statistical analyses were done usingGraphPad Prism software.

Results Computationally Optimized Broadly Reactive Antigen Design

To address the challenge of antigenic diversity present in H5N1influenza, a computationally optimized broadly reactive antigen (COBRA)was designed. For the first step of antigen generation, 129 uniquehemagglutinin (HA) sequences were downloaded from the NCBI InfluenzaVirus Resource (IVR) sequence database (Bao et al., J Virol 82:596-601,2008) representing clade 2 H5N1 viruses isolated from human infectionsbetween 2004 and 2006. The sequences were first grouped intophylogenetic sub-clades and then further divided into individualoutbreak groups within each sub-clade based upon geographic location andtime of isolation. HA amino acid sequences for each individual outbreakgroup were aligned and the most common amino acid at each position wasdetermined resulting in primary consensus sequences representing eachoutbreak group within each sub-clade (FIG. 1A). Primary consensussequences within each sub-clade were then aligned and the most commonamino acid was chosen resulting in secondary consensus sequencesrepresenting each sub-clade (FIG. 1A). The secondary consensus sequenceswere aligned and the most common amino acid at each position wasselected resulting in the final consensus sequence referred to as clade2 COBRA HA (FIG. 1A). Phylogenetic analysis of the COBRA HA with allhuman isolates of H5N1 HA proteins indicated that COBRA retained a clade2-like sequence without being grouped specifically within any clade 2sub-clade cluster (FIG. 1B). Furthermore, a BLAST search using the COBRAHA sequence revealed that it is a unique sequence that has not beenisolated from the environment.

Characterization of COBRA

Since COBRA is a fully synthetic protein, the retention of naturalhemagglutinin function was confirmed. Initially, COBRA expression wasverified by transient transfection of mammalian cells. Analysis of thetotal cell lysate demonstrated that the COBRA HA migrates at itspredicted molecular weight of approximately 73 kDa (FIG. 2A). Becausethe poly-basic cleavage site was retained in the COBRA HA sequence, bothHA0 and the HAI subunits were detected by immunoblot at similarmolecular weights as recombinant HA and the HA on the H5N1 virion (FIG.2A). Virus-like particles (VLPs) with COBRA HA on the surface boundsialic acid in a dose-dependent manner and this binding was specific toCOBRA, since empty lentiviral core alone did not bind to the receptor(FIG. 2B).

To determine if the COBRA HA was functional, the protein was pseudotypedonto lentiviral Gag_(p24) to generate pseudoparticles (Nefkens et al., JClin Virol 39(1):27-33, 2007; Haynes et al., Vaccine 27(4):530-541,2009). COBRA HA containing pseudoparticles mediated cell fusion asefficiently as H5N1 control pseudoparticles without the requirement fortrypsin. In contrast, H1N1 pseudoparticles did require trypsin andpseudoparticles without surface HA produced luciferase at similar levelsas the cell only controls (FIG. 2C). Taken together, these resultsdemonstrate that although the COBRA HA is a synthetic protein not foundin nature, it retains all of the functions of a natural hemagglutininprotein.

Mouse Dosing Immunizations

Mice (BALB/c; n=5) were vaccinated (week 0 and 3) via intramuscularinjection with purified COBRA VLPs at either a high dose (1.5 μg HA) orlow dose (0.3 μg HA) with and without Imject® alum adjuvant. At week 5,all COBRA VLP-vaccinated animals had anti-HA antibodies that recognizedheterologous recombinant HA derived from both clade 1 and alsosub-clades of clade 2 (FIGS. 3A and 3B). Imject® alum significantlyincreased anti-HA antibody titers in both low and high dose groups ascompared to the non-adjuvanted groups (p<0.01). The IgG isotypesubclasses elicited by the VLP vaccines against a clade 2.1 coatingantigen consisted mainly of IgG1 and IgG2a, indicating a mixed T helperresponse (FIGS. 3C and 3D). Similar results were found for additionalcoating antigens representing clade 1, clade 2.2 and clade 2.3. Inaddition to recognizing HA, antibodies were also evaluated for theability to block virus from binding its receptor via inhibition ofviral-induced agglutination of horse erythrocytes (HAI). All miceadministered Imject® alum adjuvanted vaccines, regardless of dose, hadHAI titers ≧1:40 to viruses expressing HA from clades 2.1 and 2.2 and90% of the mice had titers ≧1:40 to a clade 2.3 representative virus(FIGS. 3E and 3F). Non-adjuvanted vaccines elicited generally lower HAIantibody titers with 100% of high dose animals achieving titers ≧1:40only against clade 2.1 viruses. Imject® alum adjuvanted vaccineselicited significantly higher HAI antibody titers to clade 2.2 and clade2.3 viruses regardless of dose as compared to non-adjuvanted vaccines(p<0.05 for high dose and p<0.001 for low dose, respectively). None ofthe vaccines elicited high HAI titer antibodies to a clade 1 virus.

Mouse Dosing Challenge

Mice that received the COBRA VLP vaccines or mock vaccinated controlmice were challenged intranasally with a lethal dose of clade 2.2 H5N1highly pathogenic avian influenza (A/Mongolia/whooper swan/244/2005) toevaluate the protective efficacy of the different COBRA vaccineformulations. All COBRA vaccinated mice, regardless of dose or thepresence of adjuvant, were protected from weight loss and deathfollowing lethal challenge, while all mock vaccinated animals rapidlylost weight and required euthanasia by day 6 post infection (FIGS. 4Aand 4B). Additionally, COBRA VLP vaccinated mice had no signs ofdisease, while mock vaccinated animals developed such symptoms asruffled fur, hunched back, and lethargy (FIGS. 4C and 4D).

Mouse Comparison Immunizations

To determine if the COBRA HA vaccine elicits a broader antibody responsecompared to a vaccine derived from a primary isolate, an additional setof mice were vaccinated with either COBRA VLPs or clade 2.2(A/Mongolia/whooper swan/244/2005) VLPs. Mice (BALB/c; n=20) werevaccinated (week 0 and 3) via intramuscular injection with either COBRAVLPs or clade 2.2 VLPs at a high dose (3 μg HA) with Imject® alumadjuvant. At week 5, all COBRA VLP-vaccinated mice and all clade 2.2VLP-vaccinated mice had anti-HA antibodies that recognized heterologousrecombinant HA derived from both clade 1 and various sub-clades of clade2 (FIG. 5A). Although no significant differences were found in total IgGtiters between vaccine groups, COBRA VLP-vaccinated animals had higherHAI antibody titers against all viruses tested as compared to clade 2.2VLP-vaccinated animals (p<0.01; FIG. 5B). Furthermore, COBRAVLP-vaccinated animals had an increased frequency of HAI titers of ≧1:40compared to clade 2.2 VLP-vaccinated animals (Table 2).

TABLE 2 Mouse seroconversion frequency Vaccine Antigen Clade 1^(a) Clade2.1^(b) Clade 2.2^(c) Clade 2.3^(d) COBRA 45% (9/20) 100% 100% (20/20)100% (20/20) (20/20) Clade 2.2^(c)  0% (0/20) 0% (0/20)  0% (0/20)  0%(0/20) Percentage of VLP-vaccinated animals achieving an HAI titer of≧1:40 to each test antigen. ^(a)A/Vietnam/1203/2004^(b)A/Indonesia/5/2005 ^(c)A/Whooper Swan/Mongolia/244/2005^(d)A/Anhui/1/2005

Mouse Comparison Challenge

Mice that received the COBRA VLP vaccine, clade 2.2 VLP vaccine or mockvaccinated control mice were challenged intranasally with a lethal doseof clade 2.2 H5N1 highly pathogenic avian influenza (A/Mongolia/whooperswan/244/2005) to evaluate the protective efficacy of the VLP vaccines.All VLP-vaccinated mice were protected from weight loss and deathfollowing lethal challenge while all mock vaccinated animals rapidlylost weight and required euthanasia by day 6 post infection (FIG. 6A).Additionally, VLP vaccinated mice did not show signs of disease, whilemock vaccinated animals developed ruffled fur, hunched back, andlethargy (FIG. 6B). Even though the clade 2.2 VLP was matched to thechallenge virus, no significant differences were found between COBRA VLPand clade 2.2 VLP vaccinated mice in any of the parameters analyzedindicating that the COBRA VLP vaccine protected animals as efficientlyas the homologous vaccine.

Ferret Comparison Immunizations

Ferrets are the most relevant model for influenza disease and as suchthe COBRA vaccine was tested in this more rigorous animal model. Ferrets(Fitch; n=9) were vaccinated (week 0 and 3) via intramuscular injectionwith COBRA VLPs or clade 2.2 VLPs at a high dose (15 μg HA) with Imject®alum adjuvant. Serum was collected from ferrets at week 5 and antibodyresponses to the COBRA vaccines were evaluated. All vaccinated ferretshad anti-HA antibodies that recognized heterologous recombinant HAderived from both clade 1 and also sub-clades of clade 2 (FIG. 7A). Nosignificant difference in anti-HA antibody was found between the COBRAVLP vaccine and the clade 2.2 VLP vaccine for any of the antigens tested(p>0.05). In addition to recognizing HA, antibodies were also evaluatedfor HAI activity. COBRA VLP-vaccinated animals had higher HAI antibodytiters against clade 2.1 and clade 2.3 viruses as compared to clade 2.2VLP-vaccinated animals (p<0.01 FIG. 7B). Similar to the mice, COBRAVLP-vaccinated ferrets displayed an increased rate of achieving HAItiters ≧1:40 when compared to clade 2.2 VLP-vaccinated ferrets (Table3).

TABLE 3 Ferret seroconversion frequency Vaccine Antigen Clade 1^(a)Clade 2.1^(b) Clade 2.2^(c) Clade 2.3^(d) COBRA 0% (0/9) 78% (7/9) 56%(5/9) 56% (5/9) Clade 2.2^(c) 0% (0/9)  0% (0/9) 22% (2/9)  0% (0/9)Percentage of VLP-vaccinated animals achieving an HAI titer of ≧1:40 toeach test antigen. ^(a)A/Vietnam/1203/2004 ^(b)A/Indonesia/5/2005^(c)A/Whooper Swan/Mongolia/244/2005 ^(d)A/Anhui/1/2005

Ferret Comparison Challenge

Ferrets that received the COBRA VLP vaccines, clade 2.2 VLP vaccines ormock vaccinated control animals were challenged intranasally with clade2.2 H5N1 highly pathogenic avian influenza (A/Mongolia/whooperswan/244/2005) to evaluate the protective efficacy of the COBRA vaccinein the ferret model of influenza infection. All VLP vaccinated ferretswere protected from weight loss and death following viral challenge,while all mock vaccinated animals rapidly lost weight and 78% (7/9) ofmock vaccinated animals required euthanasia by day 7 post-infection(FIGS. 8A and 8B). Additionally, both COBRA VLP-vaccinated and clade2.2-vaccinated ferrets were protected from acute fever and failed todevelop significant signs of disease while mock vaccinated animals hadan elevated body temperature and developed such symptoms as lethargy,diarrhea and decreased food and water intake (FIGS. 8C and 8D). Inaddition to monitoring outward signs of disease progression, nasalwashes were collected for determination of viral replication in theupper respiratory tract. Ferrets vaccinated with either COBRA VLPs orclade 2.2 VLPs did not have detectable virus at any point afterinfection, while mock vaccinated animals had high levels of viralreplication for the first five days of the infection (FIG. 8E). Nosignificant differences were found between COBRA VLP and clade 2.2 VLPvaccinated ferrets in any of the challenge parameters analyzedconfirming the findings in mice that the COBRA VLP vaccine protectedanimals as efficiently as the homologous vaccine.

The percent identity of COBRA HA and the test antigens used in the mouseand ferret studies described above are shown in Table 4.

TABLE 4 Percent Identity of Test Antigens Vaccine Antigen Clade 1^(a)Clade 2.1^(b) Clade 2.2^(c) Clade 2.3^(d) COBRA 97% 97% 95% 97% Clade2.2^(c) 94% 97% 100% 94% HA amino acid sequences were aligned andpercent identity across the entire protein was determined for thevaccine immunogens compared to the representative test antigens.^(a)A/Vietnam/1203/2004 ^(b)A/Indonesia/5/2005 ^(c)A/WhooperSwan/Mongolia/244/2005 ^(d)A/Anhui/1/2005

Example 2 A Computationally-Optimized HA VLP Vaccines ElicitsBroadly-Reactive Antibodies that Protect Monkeys from H5N1 Infection

This example describes the finding that a COBRA clade 2 HA H5N1 VLPelicits broad humoral immunity against multiple H5N 1 isolates fromdifferent clades.

Materials and Methods Expression and Purification of Virus-LikeParticles

The COBRA HA sequence is described above in Example 1. 293T cells weretransiently transfected with plasmids expressing HA, M1, and NA in lowserum media, incubated for 72 h at 37° C., and purified byultracentrifugation through a 20% glycerol cushion as previouslydescribed (Giles and Ross, Vaccine 29:3043-3054, 2011). All VLP vaccineswere engineered using the same NA from A/Thailand/1(KAN-1)/2004. HAcontent was quantified as previously described (Giles and Ross, Vaccine29:3043-3054, 2011). Two different VLP preparations were purified, eachcontaining one of the HA influenza gene products: WS/05 or the COBRA HA.

Primate Immunizations and H5N1 Challenges

Cynomolgus macaques (Macaca fascicularis, male, 3-5 years old) werevaccinated with 15 μg (based upon HA content) of purified COBRA HA VLPs(n=7) or WS/05 VLPs (n=7), via intramuscular injection at weeks 0, 3 and6. Vaccines at each dose were formulated with alum adjuvant (Imject®Alum, Pierce Biotechnology; Rockford, Ill., USA) immediately prior touse. Twenty-one days after each vaccination, blood was collected fromanesthetized macaques. All procedures were in accordance with the NRCGuide for the Care and Use of Laboratory Animals.

Three weeks after final vaccination, macaques were placed into BSL3+isolator units (Bioqual, Inc. Rockville, Md.) and then challenged by amulti-route of infection (ocular, nasal, tracheal) as previouslydescribed (Kobasa et al., Nature 445:319-323, 2007; Kuiken et al., VetPathol 40:304-310, 2003; Rimmelzwaan et al., Avian Dis 47:931-933, 2003)using 1×10⁶ plaque forming units (PFU) of the highly pathogenic H5N1virus, A/Whooper Swan/Mongolia/244/2005 (clade 2.2), at each location.Monkeys were monitored daily for weight loss, signs of disease, andmortality until 7 days after infection. Individual body weights,sickness scores (based upon lethargy, temperature change, nasaldischarge, lack of appetite, dehydration, lack of responsiveness), anddeath were recorded for each group.

Nasal and tracheal washes were performed at day 0, 1, 3, 5, and 7post-infection. In addition, subsets of monkeys were sacrificedfollowing administration of anesthesia and necropsies were performedaccording to standard procedures for assessment of gross pathologic andhistopathologic changes, as well as the extent of virus replication.

Serological Assays

A quantitative ELISA was performed to assess anti-HA specific IgG inimmune serum as previously described (Bright et al., PLoS One 3:e1501,2008; Giles and Ross, Vaccine 29:3043-3054, 2011). The hemagglutinationinhibition (HAI) assay was used on sera treated with receptor destroyingenzyme (RDE; Denka Seiken, Co., Japan) prior to being tested (Bright etal., Vaccine 25:3871-3878, 2007; Mitchell et al., Vaccine 21:902-914,2003; Bright et al., PLoS One 3:e1501, 2008) to assess functionalantibodies to the HA able to inhibit agglutination of horse red blood(Askonas B, McMichael A, Webster R. The immune response to influenzaviruses and the problem of protection against infection. In: Beare A S,editor. Basic and applied influenza research: CRC Press 1982: 159-188).The protocol was adapted from the CDC laboratory-based influenzasurveillance manual and performed as previously described (Gillim-Rossand Subbarao, Clin Microbiol Rev 19:614 -636, 2006; Bright et al., PLoSOne 3:e1501, 2008). The HAI titer was determined by the reciprocaldilution of the last row which contained non-agglutinated RBC. Positiveand negative serum controls were included for each plate. All monkeyswere negative (HAI≦1:10) for pre-existing antibodies to currentlycirculating human influenza viruses prior to vaccination. Serumneutralizing antibody titers were determined by microneutralization (MN)assays performed on Madin Darby Canine Kidney (MDCK) cells following theprocedure until CPE was observed (Rowe et al., J Clin Microbiol37:937-943, 1999). Cells were then fixed in 10% formalin and stainedwith 1% crystal violet to quantify CPE. The neutralizing antibody titersare expressed as the reciprocal of the highest dilution of serum thatgave 50% neutralization of 100 TCID₅₀ of virus in MDCK cells. Geometricmean neutralizing antibody titers were calculated for each group.

Histopathologic Evaluation and Immunohistochemical Analysis

Formalin-inflated lungs and trachea were fixed in 10% neutral bufferedformalin. Cross-sections of upper and lower left and right lung lobesand trachea were made, concentrating on gross-lesions. Tissue wasparaffin-embedded and 6-sections were stained with hematoxylin and eosinfor histologic evaluation. Sequential sections were processed forimmunohistochemistry or in situ hybridization (ISH).Immunohistochemistry was performed as described previously (Bissel etal., Am J Pathol 160:927-941, 2002) using an immunoperoxidase methodwith a polyclonal antibody (Maine Biotechnology Services, Portland, Me.)directed against influenza A virus. A biotinylated donkey anti-goat IgG(Rockland Immunochemicals, Gilbertsville, Pa.) was used as the secondaryantibody. ISH was performed as described previously (Fallert et al., JVirol Methods 99:23-32, 2002) using a 35S-labeled riboprobe synthesizedusing templates derived from 760 bp of influenza A/California/04/2009matrix protein.

Results Vaccine Induced Antibody Responses

Cynomolgus macaques were vaccinated with COBRA VLPs or WS/05 VLPsformulated with Imject® alum at 0, 3 and 6 weeks. At week 3post-vaccination, all COBRA VLP-vaccinated animals had anti-HAantibodies that recognized recombinant HA derived from three sub-cladesof clade 2, which were boosted at week 6 (FIGS. 10A and 10B). There wasno statistical difference (p>0.05) in the anti-HA titers elicitedagainst any of the HA proteins, except monkeys vaccinated with COBRAVLPs had a statistically higher titer against the Indo/05 HA (clade 2.1)compared with monkeys vaccinated with the WS/05 VLP (derived from clade2.2) on week 6.

A Single COBRA VLP Vaccination Induced High Titer HAI and MN Antibodiesto Clade 2 H5N1 Viruses

Monkeys vaccinated with COBRA VLPs (but not with WS/05 VLPs) had HAIactivity against viruses representing all three clade 2 sub-clades aftera single vaccination (FIG. 10C). Four to six monkeys responded to theCOBRA VLP vaccine with an HAI titer ≧1:40 for the all of the varioustest antigens. In contrast, 4 of 7 monkeys vaccinated with the WS/05 VLPresponded to the homologous clade 2.2 virus, but none of thesevaccinated monkeys responded to the clade 2.1 or 2.3 virus. Following asecond vaccination, almost all the monkeys vaccinated with eithervaccine responded to all three viruses (FIG. 11D). These results wereconfirmed by microneutralization assay (FIGS. 11E and 11F). However,monkeys vaccinated with COBRA VLPs showed boosted HAI titers to allthree clade 2 viruses (FIG. 11).

COBRA VLPs Induced HAI Antibodies that Recognize Broader Numbers of H5N1Viruses

In order to determine if the COBRA HA elicited antibodies thatrecognized a broader number of H5N1 isolates, serum was collected andtested for the ability to inhibit influenza virus inducedhemagglutination of red blood cells in vitro. Antisera collected fromboth vaccinated and unvaccinated monkeys were then tested against abroad panel of H5N1 viruses representing not only sub-clades of clade 2,but also non-clade 2 H5N1 virus strains (0, 1, 4, and 7) by HAI. Monkeysvaccinated with the COBRA VLP had high average HAI titers against allclade 2 isolates, regardless of sub-clade (FIG. 11). In general, all 7monkeys responded to the COBRA VLP vaccine and seroconverted with an HAItiter ≧1:40 against all the clade 2 viruses. In contrast, monkeysvaccinated with the WS/05 VLP vaccine had lower HAI titers against clade2 viruses (FIG. 10) and fewer number of monkeys responded to thevaccine. Of the 10 clade 2 viruses tested in the HAI assay, WS/05 VLPvaccinated monkeys responded more poorly (fewer than 4 of 7 monkeys) to4 of the isolates and none of these monkeys had antibodies thatresponded to the Dk/HU/02 (clade 2.1.1) or Eg/3300/08 (clade 2.2.1)isolates. The COBRA VLPs elicited significantly higher HAI titersagainst almost all of the clade 2 viruses than the WS/05 VLPs (FIG. 11).

In addition to clade 2 isolates, a minimum of five COBRA VLP vaccinatedmonkeys had HAI antibodies against both clade 1 and 7 virus isolates(FIG. 11). In comparison, almost none of the WS/05 VLP vaccinatedmonkeys had HAI antibodies against clade 1 and clade 7 viruses. None ofthe monkeys, regardless of the vaccine, had antibodies that responded tothe clade 0 or 4 isolates. All mock vaccinated monkeys did not recognizeany of the H5N1 isolates.

Challenge of Vaccinated and Unvaccinated Primates with H5N1 Clade 2.2Virus

Three weeks after final vaccination, both VLP vaccinated andmock-vaccinated monkeys were transferred to ABSL3+ isolator units andthen challenged with highly pathogenic H5N1 virus, A/WhooperSwan/Mongolia/244/2005 (clade 2.2) (1×10⁶ pfu), by a multi-route(ocular, nasal, tracheal, oral) of infection (Kobasa et al., Nature445:319-323, 2007; Kuiken et al., Vet Pathol 40:304-310, 2003;Rimmelzwaan et al., Avian Dis 47:931-933, 2003). There was nosignificant weight loss or mortality in any of the monkeys over the 7day period of observation. Unvaccinated monkeys had an elevatedtemperature of ˜2° C. that was sustained for 5 days post-infection andhigher gross pathology scores by day 3 post-infection (Table 5).

TABLE 5 Lung pathology, temperature and viral titer of vaccinatedmacaques Lung Pathology Elevated Peak Viral Titer Vaccine Score (day 3)temperature (days) (pfu/ml) (day) Mock 5.3 1.9° C. (1-5 DPI) Nasal wash:2.2-2.5 (5 DPI) Trachea wash: 2.0-4.4 (3 DPI) WS/05 VLP 3.3 1.1° C.-1.3°C. Nasal wash: <2 (1-5 DPI) Trachea wash: <2 COBRA 2.1 1.3° C. (2 DPI)Nasal wash: <2 VLP Trachea wash: <2

The lungs of unvaccinated monkeys had mild to moderate acute pneumoniawith alveolar pulmonary exudate by day 3 post-infection by H&E staining.ISH showed focal collections of H5N1 infected cells present at day 3post-infection in alveolar spaces and to a lesser extent in bronchialepithelium. These results were similar to unvaccinated monkeys infectedwith the clade 1 H5N1 virus, A/Vietnam/1203/2004. In contrast, monkeysvaccinated with either the COBRA VLP or the WS/05 VLP vaccine had areduced gross pathology scores of 2.1-3.3 at day 3 post-infection with amilder increase in body temperature (1.1-1.3° C.) that spiked betweendays 2-3 post-infection and then returned to pre-infection temperatures.Vaccinated animals had fewer H5N1 infected cells that were detectedprimarily on day 1 post-infection (Table 6).

TABLE 6 H5N1 lung infection scores Alveolar Submucosal infectioninfection Vaccine score score 1 3 5 1 3 5 1 3 5 Mock 1.00 0.05 0 1.100.48 0.25 0 0 0 WS/05 VLP 0.05 0 0 0.55 0.10 0 0 0 0 COBRA 0 0 0 0.600.03 0.05 0 0 0 VLP ISH for influenza was performed on tissue sectionsof from upper and lower left and right lung. A semi-quantitative scoringsystem was developed to evaluate the presence of influenza infectedcells. Scores were then averaged: 0.2 = rare or occasional cells but <5%of fields; 1 = >½ to ¼ low power fields; 2 = >¼ low power fields; 3 =essentially all low power fields.

However, monkeys vaccinated with the COBRA VLP had little to no signs oflung inflammation by H&E staining, while animals vaccinated with theWS/05 VLP vaccine had similar signs of inflammation as non-vaccinatedmonkeys (Table 7). In addition, unvaccinated monkeys had high titers ofvirus in both the nasal and tracheal washes between days 3 and 5post-infection. In contrast, no virus was detected in either vaccinatedgroups.

TABLE 7 Lung involvement and inflammation scores % lung BronchialAlveolar involvement^(a) inflammation^(b) inflammation^(b) Vaccine 1 3 51 3 5 1 3 5 Mock 0.38 1.13 1.25 0.63 0.75 1.25 0.63 1.00 1.25 (0-1)(0-2) (0-2) (0-1) (0-2) (0-2) (0-1) (0-2) (0-2) WS/05 0.75 1.50 0.881.00 1.42 0.63 1.00 1.25 1.00 VLP (0-2) (0-3) (0-3) (1)   (1-2) (0-2)(0-2) (0-2) (0-2) CO- 0.88 0.50 0.38 1.13 0.75 0.88 1.13 0.67 0.25 BRA(0-2) (0-2) (0-2) (1-2) (0-2) (0-2) (0-2) (0-2) (0-1) VLP ^(a)% Lunginvolvement. Tissue sections from upper and lower left and right lungwere evaluated for percent area demonstrating pneumonia. Scores werethen averaged. Range in parentheses. 0 = <10%, 1 = 10-24%, 2 = 25-50%, 3= >50%. ^(b)Bronchial and alveolar inflammation scores. Tissue sectionsfrom upper and lower left and right lung were evaluated for presence ofbronchial inflammation and denudation and alveolar immune cellinfiltration. Scores were then averages: 0 = absent, 1 = present, 2 =abundant.

Example 3 Comparison of Protective Efficacy by Vaccination withComputationally Optimized HA and Polyvalent HA Based H5N1 VLP Vaccines

This example describes a comparison of the COBRA HA vaccine to apolyvalent H5N1 vaccine. The results demonstrate that a single COBRAantigen elicits broader antibodies and is more effective than apolyvalent mixture of primary antigens.

Materials and Methods Vaccine Antigens and Preparation

The design and characterization of the computationally optimized broadlyreactive antigen (COBRA) is described in Example 1. Polyvalent vaccineHA antigens were derived via reverse transcription from the following6:2 reassortant H5N1 viruses: A/Indonesia/5/2005 (clade 2.1; IN/05),A/Whooper Swan/Mongolia/244/2005 (clade 2.2; WS/05) and A/Anhui/1/2005(clade 2.3; AN/05). All HA antigens were cloned into the pTR600expression vector. Virus-like particles (VLPs) were generated bytransiently transfecting HEK 293T cells with plasmids expressing M1(A/Puerto Rico/8/1934), NA (A/Thailand/1(KAN-1)/2004), and a single HAfor each preparation. Cells were incubated for 72 h at 37° C. afterwhich supernatants were harvested. Cell debris was cleared by low speedcentrifugation followed by vacuum filtration through a 0.22 jam sterilefilter. VLPs were purified by ultracentrifugation (100,000×g through 20%glycerol, weight to volume) for 4 hours at 4° C. Pellets were thenresuspended in PBS pH 7.2 and stored in single use aliquots at −80° C.until use. Total protein concentration was determined by MicroBCA™Protein Assay Reagent Kit (Pierce Biotechnology, Rockford, Ill., USA).HA specific content of each VLP was determined by scanning densitometryas described previously (Giles and Ross, Vaccine 29:3043-3054, 2011).Briefly, purified HA matched to each VLP was electrophoresed withpurified VLPs, transferred to a PVDF membrane and probed by western blotwith H5-specific antisera. The relative density of the HA band in thepurified protein lanes was used to calculate a standard curve and thedensity of the HA in the VLP lanes was interpolated. In total, fourdifferent VLP preparations were purified and HA content quantifiedindependently, each containing one of the three wild-type influenza geneproducts (1N/05, WS/05, AN/05) or the COBRA HA.

Mouse Studies

BALB/c mice (Mus musculis, females, 6-8 weeks) were purchased fromHarlan Sprague Dawley, (Indianapolis, Ind., USA) and housed inmicroisolator units and allowed free access to food and water and werecared for under USDA guidelines for laboratory animals. Mice werevaccinated with purified COBRA VLPs (3 μg HA) or a polyvalentformulation of VLPs consisting of 1 μg HA each 1N/05, WS/05 and AN/05 (3μg HA total) via intramuscular injection at week 0 and then boosted atweek 3. Vaccines were formulated with Imject® alum adjuvant (Imject®Alum, Pierce Biotechnology; Rockford, Ill., USA) according to themanufacturer's protocol. Fourteen to twenty-one days after eachvaccination, blood was collected from anesthetized mice via theretro-orbital plexus and transferred to a microfuge tube. Tubes werecentrifuged and sera was removed and frozen at −20±5° C.

Three weeks after final vaccination, mice were challenged intranasallywith 5×10³ plaque forming units (PFU) of either highly pathogenic wildtype H5N1 virus A/Whooper Swan/Mongolia/244/2005 (n=20/group) or 6:2reassortant virus with internal genes from the mouse adapted virusA/Puerto Rico/8/1934 and the surface proteins HA and NA fromA/Vietnam/1203/2004 (n=10/group) in a total volume of Challenge dosesfor both viruses were established independently and representapproximately 50 LD₅₀. After infection, mice were monitored daily forweight loss, disease signs and death for 14 days after infection.Individual body weights, sickness scores and death were recorded foreach group on each day after inoculation. Sickness score was determinedby evaluating activity (0=normal, 1=reduced, 2=severely reduced),hunched back (0=absent, 1=present) and ruffled fur (0=absent, 1=present)(Toapanta and Ross, Respiratory Res 10(1):112, 2009). Experimentalendpoint was determined by >20% weight loss or display of neurologicaldisease such as hind limb paralysis. All highly pathogenic wild typeH5N1 influenza virus studies were performed under high-containmentbiosafety level 3 enhanced conditions (BSL3+).

Ferret Studies

Fitch ferrets (Mustela putorius faro, female, 6-12-months of age),influenza naïve and descented, were purchased from Marshall Farms(Sayre, Pa., USA). Ferrets were pair housed in stainless steel cages(Shor-line, Kansas City, Kans., USA) containing Sani-chips LaboratoryAnimal Bedding (P.J. Murphy Forest Products, Montville, N.J., USA).Ferrets were provided with Teklad Global Ferret Diet (Harlan Teklad,Madison, Wis., USA) and fresh water ad libitum. The VLPs were diluted inPBS, pH 7.2 to achieve final concentration. Ferrets (n=6) werevaccinated with purified COBRA VLPs (15 μg HA) or a polyvalentformulation of VLPs consisting of 5 μg HA each IN/05, WS/05 and AN/05(15 μg HA total) via intramuscular injection at week 0 and then boostedat week 3. Vaccines were formulated with Imject® alum adjuvant (Imject®Alum, Pierce Biotechnology; Rockford, Ill., USA) immediately prior touse according to the manufacturer's protocol. Animals were monitored foradverse events including weight loss, temperature, loss of activity,nasal discharge, sneezing and diarrhea weekly during the vaccinationregimen. Prior to vaccination, animals were confirmed by HAI assay to beseronegative for circulating influenza A (H1N1 and H3N2) and influenza Bviruses. Fourteen to twenty-one days after each vaccination, blood wascollected from anesthetized ferrets via the anterior vena cava andtransferred to a microfuge tube. Tubes were centrifuged and sera wasremoved and frozen at −20±5° C.

Three weeks after final vaccination, ferrets were challengedintranasally with 1×10⁶ plaque forming units (PFU) of the highlypathogenic H5N1 virus A/Whooper Swan/Mongolia/244/2005 (clade 2.2) in avolume of 0.5 ml in each nostril for a total infection volume of 1 ml.After infection, ferrets were monitored daily for weight loss, diseasesigns and death for 14 days after infection. Individual body weights,sickness scores, and death were recorded for each group on each dayafter inoculation. Sickness score was determined by evaluating activity(0=normal, 1=alert and active after stimulation, 2=alert but not activeafter stimulation, 3=neither active nor alert after stimulation), nasaldischarge (0=absent, 1=present), sneezing (0=absent, 1=present),decreased food intake (0=absent, 1=present), diarrhea (0=absent,1=present), dyspnea (0=absent, 1=present) and neurological symptoms(0=absent, 1=present) as previously described (Giles and Ross, Vaccine29:3043-3054, 2011). Experimental endpoint was defined as >20% weightloss, development of neurological disease or an activity score of 3(neither active nor alert after stimulation). Nasal washes wereperformed by instilling 3 ml of PBS into the nares of anesthetizedferrets each day for 14 days after inoculation. Washes were collectedand stored at −80° C. until use. All highly pathogenic wild type H5N1influenza virus studies were performed under high-containment biosafetylevel 3 enhanced conditions (BSL3+).

ELISA Assay

The ELISA assay was used to assess total antibody titer to the HA. Highbinding, 96-well polystyrene plates (Costar; Lowell, Mass., USA) werecoated overnight with 50 ng/well of recombinant HA. Coating antigenswere derived from the following representative viral isolates:A/Vietnam/1203/2004 (clade 1), A/Indonesia/5/2005 (clade 2.1), A/WhooperSwan/Mongolia/244/2005 (clade 2.2) and A/Anhui/1/2005 (clade 2.3).Plates were blocked with 5% milk diluted in PBS with 0.05% Tween 20.Serum samples were diluted in blocking buffer and added to plates. Serumwas two-fold serially diluted and allowed to incubate for 1 hour at roomtemperature. Plates were washed and species specific antibody againstIgG linked to horseradish peroxidase (HRP) was diluted in blockingbuffer and added to plates. Plates were incubated for 1 hour at roomtemperature. Plates were washed and HRP was developed with TMB substrate(Sigma-Aldrich; St. Louis, Mo., USA). Plates were incubated in the darkfor 15 minutes and then the reaction was stopped with 2N H₂SO₄. Opticaldensities at a wavelength of 450 nm (OD₄₅₀) were read by aspectrophotometer (BioTek; Winooski, Vt., USA) and end point dilutiontiters were determined as the reciprocal dilution of the last well whichhad an OD₄₅₀ above the mean OD₄₅₀ plus two standard deviations of naïveanimal sera.

Hemagglutination Inhibition (HAI) Assay

The HAI assay was used to assess functional antibodies to the HA able toinhibit agglutination of horse erythrocytes. The protocol was adaptedfrom the CDC laboratory-based influenza surveillance manual (Gillim-Rossand Subbarao, Clin Microbiol Rev 19(4):614-636, 2006). To inactivatenon-specific inhibitors, sera were treated with receptor destroyingenzyme (RDE; Denka Seiken, Co., Japan) prior to being tested. Briefly,three parts RDE was added to one part sera and incubated overnight at37° C. RDE was inactivated by incubation at 56° C. for ˜30 min. RDEtreated sera was two-fold serially diluted in v-bottom microtiterplates. An equal volume of reassortant virus, adjusted to approximately8 HAU/50 μl, was added to each well. The reassortant viruses containedthe internal genes from the mouse adapted strain A/Puerto Rico/8/1934and the surface proteins HA and NA from the following representativeviral isolates: A/Vietnam/1203/2004 (clade 1), A/Indonesia/5/2005 (clade2.1), A/Whooper Swan/Mongolia/244/2005 (clade 2.2) and A/Anhui/1/2005(clade 2.3). The plates were covered and incubated at room temperaturefor 20 minutes followed by the addition of 1% horse erythrocytes (HRBC)(Lampire Biologicals, Pipersville, Pa., USA) in PBS. Red blood cellswere stored at 4° C. and used within 72 h of preparation. The plateswere mixed by agitation, covered, and the RBCs were allowed to settlefor 1 h at room temperature (Askonas B, McMichael A, Webster R. Theimmune response to influenza viruses and the problem of protectionagainst infection. In: Beare A S, editor. Basic and applied influenzaresearch: CRC Press 1982: 159-188). The HAI titer was determined by thereciprocal dilution of the last well which contained non-agglutinatedRBC. Positive and negative serum controls were included for each plate.All mice and ferrets were negative (HAI≦1:10) for pre-existingantibodies to currently circulating human influenza viruses prior tovaccination.

Plaque Assay

For mouse infections, lung virus titers were evaluated. For ferretinfections, nasal wash virus titers were used to assess viral burden.Both lungs and nasal wash virus titers were determined using a plaqueassay (Tobita et al., Med Microbiol Immunol 162:23-27, 1975; Tobita etal., Med Microbial Immunol 162:9-14, 1975). Briefly, lungs from miceinfected with virus were harvested post infection, snap-frozen andstored at −80° C. until use. Samples were thawed, weighed and singlecell suspensions were prepared via passage through a 70 μm mesh (BDFalcon, Bedford, Mass., USA) in an appropriate volume of DMEMsupplemented with penicillin-streptomycin (iDEME) as to achieve 100mg/ml final concentration. Cell suspensions were centrifuged at 2000 rpmfor 5 minutes and the supernatants were collected.

Madin-Darby Canine Kidney (MDCK) cells were plated (5×10⁵) in each wellof a 6 well plate. Samples (lung supernatants for mice and nasal washesfor ferrets) were diluted (dilution factors of 1×10¹ to 10⁶) andoverlayed onto the cells in 100 μl of iDMEM and incubated for 1 hour.Virus-containing medium was removed and replaced with 2 ml of L15 mediumplus 0.8% agarose (Cambrex, East Rutherford, N.J., USA) and incubatedfor 96 hours at 37° C. with 5% CO₂. Agarose was removed and discarded.Cells were fixed with 10% buffered formalin, and then stained with 1%crystal violet for 15 minutes. Following thorough washing in dH₂O toremove excess crystal violet, plates were allowed to dry, plaquescounted, and the plaque forming units (PFU)/g for or PFU/ml for nasalwashes were calculated.

Histopathological Analysis

Left lobes of lungs from infected mice were collected 4 dayspost-infection and placed into 10% buffered formalin. After fixation,lungs were paraffin embedded and 6 μm sections were prepared forhistopathological analysis. For in situ hybridization (ISH), vectorscontaining 760 bp of Influenza/California/04/2009 matrix protein werelinearized to create antisense and sense templates. ³⁵S-labeledriboprobes were generated using MAXIscript in vitro transcription kit(Ambion, Austin, Tex.). ISH was performed as described before (Bissel etal., Brain Pathol, Accepted Article doi:10.1111/j.1750-3639.2010.00514.x). Control riboprobes did not hybridizeto lung tissue at any time point post-infection and non-infected tissuedid not show hybridization with viral probes. Hybridized slides wereassessed and scored for abundance of foci.

Cellular Assays

The number of anti-influenza specific cells secreting interferon gamma(IFN-γ) was determined by enzyme-linked immunospot (ELISpot) assay (R&Dsystems, Minneapolis, Minn., USA) following the manufacturer's protocol.Mice were sacrificed at 6 days post infection (DPI) and spleens andlungs were harvested and prepared in single cell suspensions. Briefly,pre-coated anti-IFNγ plates were blocked with RPMI plus 10% FCS andantibiotics (cRPMI) for 30 minutes at room temperature. Media wasremoved from wells and 10⁵ cells were added to each well. Cells werestimulated with purified recombinant HA from A/Vietnam/1203/2004(truncated at residue 530; 1 μg/well), inactivated 6:2 reassortant virusA/Vietnam/1203/2004 (1:100 dilution of inactivated stock; 100 μl/well)or the immunodominant H2-K^(d) CD8⁺ T cell epitope in H5 HA: HA₅₃₃(IYSTVASSL; SEQ ID NO: 10; 1 μg/well) (Pepscan Presto, Leystad,Netherlands). Additional wells were stimulated with PMA (50 ng/well) andionomycin (500 ng/well) as positive controls or Ova₂₅₇ (SIINFEKL; SEQ IDNO: 11; 1 μg/well) (Pepscan Presto, Leystad, Netherlands) as negativecontrols. Additionally, IL-2 (10 U/ml) was added to each well. Plateswere incubated at 37° C. for 48 hours. After incubation, plates werewashed four times with R&D wash buffer and were incubated at 4° C.overnight with biotinylated anti-mouse IFNγ. Plates were washed asbefore and incubated at room temperature for 2 hours with streptavidinconjugated to alkaline phosphatase. Plates were washed as before andspots were developed by incubating at room temperature for 1 hour in thedark with BCIP/NBT chromogen substrate. The plates were washedextensively with DI H₂O and allowed to dry overnight prior to spotsbeing counted using an ImmunoSpot ELISpot reader (Cellular TechnologyLtd., Cleveland, Ohio, USA).

The number of anti-HA and anti-NA specific antibody secreting cells wasdetermined by B cell ELISpot assay as previously described (Joo et al.,Vaccine 28:2186-2194, 2009; Sasaki et al., PLoS ONE 3:e2975, 2008;Sasaki et al., J Virol 81:215-228, 2007). Mice were sacrificed at 6 DPIand spleens and lungs were harvested and prepared in single cellsuspensions. Briefly, 0.45 μm PVDF membrane plates (Millipore,Billerica, Mass., USA) were coated with either purified recombinant HAfrom A/Vietnam/1203/2004 or purified recombinant NA from A/Thailand/1(KAN-1)/2004 (250 ng/well) and incubated at 4° C. overnight. Plates werewashed three times with PBS and blocked with cRPMI for at 37° C. for 3-4hours. Media was removed from wells and 10⁵ cells were added to eachwell. Plates were incubated at 37° C. for 48 hours. After incubation,plates were washed as before and incubated at room temperature for 2hours with horse radish peroxidase conjugated anti-mouse IgG or IgA(Southern Biotech, Birmingham, Ala., USA). Plates were washed as beforeand spots were developed at room temperature for 1 hour in the dark withdetection substrate (NovaRED™; Vector Labs, Burlingame, Calif., USA).The plates were washed extensively with DI H₂O and allowed to dryovernight prior to spots being counted using an ImmunoSpot ELISpotreader (Cellular Technology Ltd., Cleveland, Ohio, USA).

Passive Transfer of Sera

Serum from vaccinated mice was pooled and passively transferred into 9week old recipient BALB/c mice (n=5/group). Equal amounts of serum fromeach mouse in a particular vaccine group were pooled and heatinactivated for 30 minutes at 56° C. 200 μl of pooled and inactivatedserum was transferred to recipient mice via IP injection. 24 hours posttransfer, mice were infected with 6:2 reassortant virus with internalgenes from the mouse adapted virus A/Puerto Rico/8/1934 and surfaceantigens from A/Vietnam/1203/2004.

Statistical Analysis

Statistical significance of the antibody and cellular immunology datawas determined using a two-tailed Student's T test to analyzedifferences between COBRA and polyvalent vaccine groups for each of thedifferent test antigens. Differences in weight loss and sickness scorewere analyzed by two-way ANOVA, followed by Bonferroni's post test foreach vaccine group at multiple time points (multiparametric).Statistical significance of viral titer data was evaluated using atwo-tailed Student's T test on Log₁₀ transformed values. Significancewas defined as p<0.05. Statistical analyses were done using GraphPadPrism software.

Results Immunogenicity in Mice and Ferrets

BALB/c mice were vaccinated twice via intramuscular injection witheither purified COBRA or polyvalent VLPs and two weeks after the secondvaccination serum was analyzed for antibody responses. All vaccinatedmice had high titer anti-HA antibodies that bound to recombinant HAderived from both clade 1 and various sub-clades of clade 2 (FIG. 12A).Although both COBRA and polyvalent vaccines elicited similar total IgG,COBRA vaccinated animals had higher HAI antibody titers for all virusestested (p<0.001; FIG. 12B). In addition to higher HAI titer, COBRAvaccinated mice had an increased frequency of HAI titers ≧1:40 for allviruses tested, including those which were components of the polyvalentformulation (Table 8).

To confirm the results from mice in a more rigorous animal model,ferrets were vaccinated twice via intramuscular injection with eitherCOBRA or polyvalent vaccines. Serum was collected two weeks after thesecond vaccination and antibody responses were evaluated. Similar to themice, all vaccinated ferrets had anti-HA antibodies that bound todiverse recombinant HA and the relative total IgG titers were equivalentfor both COBRA and polyvalent vaccines (FIG. 12C). COBRA vaccinatedferrets demonstrated increased HAI antibody titers compared topolyvalent vaccinated animals against all viruses tested, however onlythe antibodies to the clade 2.1 virus were significantly different(p<0.05; FIG. 12D). Furthermore, COBRA vaccinated animals displayed anincreased rate of achieving an HAI titer of ≧1:40 in comparison to thepolyvalent vaccinated ferrets for all test antigens (Table 8).

TABLE 8 Seroconversion frequency Vaccine Species antigen Clade 1 Clade2.1 Clade 2.2 Clade 2.3 Mouse COBRA  60% (18/30) 100% 100% 100% (30/30)(30/30) (30/30) Polyvalent  3.3% (1/30)  70%  50%  53% (21/30) (15/30)(16/30) Ferret COBRA 33% (2/6) 67% (4/6) 50% (3/6) 50% (3/6) Polyvalent 0% (0/6) 33% (2/6)  0% (0/6)  0% (0/6)

Wild Type Clade 2.2 Challenge

To confirm protective efficacy against highly pathogenic H5N1 infection,vaccinated animals were challenged with a lethal dose of the wild-typeclade 2.2 isolate A/Whooper Swan/Mongolia/244/2005. All VLP vaccinatedmice were protected from weight loss and death while mock vaccinatedanimals rapidly lost weight and reached experimental end-point by 6 dayspost infection (DPI; FIG. 13A). COBRA and polyvalent vaccinated miceboth had a mean maximum weight loss of 4% at 12 and 13 DPI,respectively. Additionally, all VLP vaccinated mice failed to developany overt signs of disease while mock vaccinated mice developed visibleillness (FIG. 13B).

Similar to the mice, all VLP vaccinated ferrets were protected fromdeath following a lethal challenge. Vaccinated ferrets demonstrated mildweight loss in response to the infection with COBRA vaccinated animalshaving mean maximum weight loss of 5.5% at 2 DPI and polyvalentvaccinated animals losing 6.8% at 3 DPI (FIG. 13C). Both groups rapidlyrecovered weight and failed to develop any significant signs of disease(FIG. 13D). Furthermore, VLP vaccinated animals did not demonstrate anytemperature spikes while mock vaccinated animals had an elevatedtemperature of ˜3° C. for 1-3 DPI.

To evaluate vaccine efficacy with a more sensitive output than morbidityand mortality, the viral burden of infected animals was also determined.Both COBRA and polyvalent vaccinated mice had reduced lung viral titersas quickly as 1 DPI when compared to mock vaccinated animals.Furthermore, COBRA vaccinated mice did not have detectable virus by 3DPI while polyvalent vaccinated mice demonstrated prolonged viralreplication with 1.8×10³ PFU/g at 3 DPI (p<0.05; FIG. 14A).Additionally, both VLP vaccines prevented extra-pulmonary spread of thevirus while mock vaccinated animals had detectable virus in both kidneyand liver by 3 DPI. Control of virus replication in ferrets was similarto that observed in mice, although complete clearance of the virus wasdelayed (FIG. 14B). All VLP vaccinated animals had decreased recovery ofvirus in nasal washes compared to mock vaccinated ferrets at alltimepoints tested (p<0.05). COBRA vaccinated animals did not havedetectable virus by 5 DPI. In contrast, virus replication did not reachundetectable levels until 9 DPI in polyvalent vaccinated ferrets.

Histopathology of Infected Lungs

To evaluate the location and severity of influenza viral antigen andviral replication, ISH for influenza A MP was scored on 3 DPI lungsections. COBRA vaccinated animals had rare bronchial epitheliuminfection (FIGS. 15A and 15B). Animals receiving polyvalent vaccines hadoccasional bronchial epithelium infection that was comparable to theCOBRA vaccinated animals (FIGS. 15A and 15B). This was in contrast tosignificant bronchial epithelium infection and replication observed inmock animals (FIGS. 15A and 15B).

Reassortant Clade 1 Challenge

Having established the clade 2.2 protective profile of both the COBRAand polyvalent vaccines, the efficacy of these vaccines against a moredivergent clade 1 challenge in mice was evaluated. COBRA and polyvalentvaccinated mice were challenged with 6:2 reassortant virus containingthe HA and NA proteins from the clade 1 virus A/Vietnam/1203/2004. AllVLP vaccinated animals were protected from weight loss and death whilemock vaccinated animals rapidly lost weight and reached experimentalendpoint by 7 DPI (FIG. 16A). Furthermore, vaccinated mice also did notdevelop any signs of disease throughout the course of the study (FIG.16B). Lungs were harvested at 3 DPI for determination of viral burden(FIG. 16C). COBRA vaccinated animals did not have detectable virus whilepolyvalent animals had 1.1×10³ PFU/g virus (p=0.12). Importantly, bothvaccines had significantly less recoverable virus than mock vaccinatedanimals at 3 DPI (p<0.01).

Post-Challenge Cellular Immune Responses

The magnitude of influenza specific cellular immune responses in thelungs post-infection was evaluated via ELISpot assay for both antibodysecreting cells (ASC) and IFN-γ producing cells. Vaccinated mice wereinfected with reassortant A/Vietnam/1203/2994 virus as before and lungswere harvested at 6 DPI. COBRA and polyvalent vaccinated animals hadstatistically equivalent numbers of both IgG and IgA ASC specific for HAfrom the challenge virus (p>0.05; FIG. 17A). No ASC were detected inmock vaccinated animals indicating that the 6 DPI time point is likelyrepresentative of a recall response. Additionally, the majority of theASC response to infection was specific for HA as lower numbers of cellswere detected for the NA component of the vaccines.

VLP vaccine primed IFN-γ secreting cells were also evaluated afterinfection. IFN-γ responses were equivalent between VLP vaccine groupsregardless of stimulating antigen (p>0.05; FIG. 17B). Recombinant HA andinactivated virus were inefficient stimulators of IFN-γ productioncompared to the HA₅₃₃ peptide. HA₅₃₃ is the immunodominant CD8′ T cellepitope in BALB/c mice and is conserved in all HA vaccine antigens usedin this study. Overlapping peptide pools spanning the entire HA moleculewere also used to stimulate cells and no differences were observedbetween COBRA and polyvalent vaccines for any of the pools. Similar tothe ASC data, no IFN-γ responses were detectable above background inmock vaccinated animals at 6 DPI.

Passive Transfer of Immune Sera

The contribution of serum factors to protection from clade 1 challengewas evaluated using a passive transfer model. Nine-week old recipientmice were administered pooled sera via IP injection from COBRA,polyvalent and mock vaccinated mice. The next day, recipient mice werechallenged with the clade 1 reassortant A/Vietnam/1203/2004 virus asbefore. Regardless of transferred serum, all recipient mice lost weightand became visibly ill (FIGS. 18A and 18B). COBRA serum recipient micelost less weight than polyvalent recipient mice with maximum losses of5.2% (6 DPI) and 11.8% (7 DPI), respectively (p<0.05 at 7 DPI). COBRAserum recipient mice also began to resolve the clinical symptoms morerapidly than polyvalent recipient mice (p<0.05 at 7 DPI). Although COBRAserum prevented recipient mice from developing illness more efficientlythan polyvalent serum, both COBRA and polyvalent serum protected allrecipient mice from death. Conversely, all mice receiving serum frommock vaccinated mice rapidly lost weight, became visibly ill and reachedexperimental endpoint by 7 DPI.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only examples of the disclosure and shouldnot be taken as limiting the scope of the invention. Rather, the scopeof the invention is defined by the following claims. We therefore claimas our invention all that comes within the scope and spirit of theseclaims.

1. An isolated nucleic acid molecule comprising a nucleotide sequenceencoding an influenza hemagglutinin (HA) polypeptide, wherein thenucleotide sequence encoding the HA polypeptide is at least at least 96%identical to SEQ ID NO:
 1. 2. The isolated nucleic acid molecule ofclaim 1, wherein the nucleotide sequence encoding the HA polypeptidecomprises SEQ ID NO:
 1. 3. (canceled)
 4. An influenza HA polypeptideencoded by the nucleic acid molecule of claim
 1. 5. A vector comprisingthe isolated nucleic acid molecule of claim
 1. 6. (canceled)
 7. Thevector of claim 5, wherein the nucleotide sequence of the vectorcomprises SEQ ID NO:
 7. 8. (canceled)
 9. An isolated cell comprising thevector of claim
 5. 10. An isolated influenza HA polypeptide, wherein theamino acid sequence of the polypeptide is at least 99% identical to SEQID NO:
 2. 11. The influenza HA polypeptide of claim 10, wherein theamino acid sequence of the polypeptide comprises SEQ ID NO:
 2. 12.(canceled)
 13. A fusion protein comprising the polypeptide of claim 10.14. An influenza virus-like particle (VLP) comprising the polypeptide ofclaim
 10. 15. The influenza VLP of claim 14, further comprising aninfluenza neuraminidase (NA) protein and an influenza matrix (M1)protein.
 16. The influenza VLP of claim 15, wherein the amino acidsequence of the influenza NA protein is at least 95% identical to SEQ IDNO: 4, the amino acid sequence of the influenza M1 protein is at least95% identical to SEQ ID NO: 6, or both. 17-26. (canceled)
 27. Acollection of plasmids comprising: (i) a plasmid encoding an influenzaNA (ii) a plasmid encoding an influenza M1; and (iii) a plasmid encodinga codon-optimized influenza HA, wherein the nucleotide sequence encodingthe codon-optimized influenza HA is at least 96% identical to SEQ IDNO:
 1. 28. The collection of claim 27, wherein the influenza NA iscodon-optimized, the influenza M1 is codon-optimized, or both.
 29. Thecollection of claim 28, wherein the nucleotide sequence encoding thecodon-optimized influenza NA is at least 95% identical to SEQ ID NO: 3,the nucleotide sequence encoding the codon-optimized influenza M1 is atleast 95% identical to SEQ ID NO: 5, or both. 30-31. (canceled)
 32. Thecollection of claim 27, wherein: (i) the plasmid encoding influenza NAcomprises SEQ ID NO: 8; (ii) the plasmid encoding influenza M1 comprisesSEQ ID NO: 9; (iii) the plasmid encoding influenza HA comprises SEQ IDNO: 10; or (iv) any combination of (i) to (iii).
 33. A compositioncomprising the influenza HA protein of claim 10 and a pharmaceuticallyacceptable carrier.
 34. A method of eliciting an immune response toinfluenza virus in a subject, comprising administering the influenza HAprotein of claim 10, thereby eliciting an immune response to influenzavirus.
 35. The method of claim 34, further comprising administering anadjuvant.
 36. (canceled)
 37. A method of immunizing a subject againstinfluenza virus, comprising administering to the subject a compositioncomprising the VLP of claim 14 and pharmaceutically acceptable carrier.38. The method of claim 37, wherein the composition further comprises anadjuvant.
 39. The method of claim 37, wherein the composition isadministered intramuscularly.
 40. The method of claim 37, wherein thecomposition comprises about 1 to about 25 μg of the VLP.
 41. (canceled)42. A method of generating an optimized influenza virus polypeptidesequence, comprising: (i) obtaining the amino acid sequences of thepolypeptide from a group of influenza virus isolates, wherein theinfluenza virus isolates are from the same subtype; (ii) organizing theamino acid sequences of the polypeptide from the group of influenzavirus isolates by clade or sub-clade and then by geographical regionwithin each clade or sub-clade; (iii) aligning the amino acid sequenceswithin each geographical region to generate primary consensus sequences,wherein each geographic region is represented by a primary consensussequence; (iv) aligning the primary consensus sequences to generatesecondary consensus sequences, wherein each clade or sub-clade isrepresented by a secondary consensus sequence; and (v) aligning thesecondary consensus sequences to generate the optimized influenza viruspolypeptide sequence.
 43. The method of claim 42, further comprising:(i) reverse translating the optimized influenza virus polypeptidesequence to generate a coding sequence; and (ii) optimizing the codingsequence for expression in mammalian cells.